Diblock copolymers and polynucleotide complexes thereof for delivery into cells

ABSTRACT

Described herein are copolymers, and methods of making and utilizing such copolymers. Such copolymers have at least two blocks: a first block that has at least one unit that is hydrophilic at physiologic pH, and a second block that has hydrophobic groups. This second block further has at least one unit with a group that is anionic at about physiologic pH. The described copolymers are disruptive of a cellular membrane, including an extracellular membrane, an intracellular membrane, a vesicle, an organelle, an endosome, a liposome, or a red blood cell. Preferably, in certain instances, the copolymer disrupts the membrane and enters the intracellular environment. In specific examples, the copolymer is endosomolytic.

CROSS-REFERENCE(S) TO RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No.15/264,392, filed Sep. 13, 2016, which is a continuation of U.S.application Ser. No. 12/992,517, filed Feb. 9, 2011, which is a U.S.National Stage entry of International Application No. PCT/US2009/043847,filed May 13, 2009, which claims the benefit of U.S. ProvisionalApplication No. 61/052,908, filed May 13, 2008, U.S. ProvisionalApplication No. 61/052,914, filed May 13, 2008, U.S. ProvisionalApplication No. 61/091,294, filed Aug. 22, 2008, U.S. ProvisionalApplication No. 61/112,054, filed Nov. 6, 2008, U.S. ProvisionalApplication No. 61/112,048, filed Nov. 6, 2008, U.S. ProvisionalApplication No. 61/120,769, filed Dec. 12, 2008, U.S. ProvisionalApplication No. 61/140,779, filed Dec. 24, 2008, U.S. ProvisionalApplication No. 61/140,774, filed Dec. 24, 2008, and U.S. ProvisionalApplication No. 61/171,377, filed Apr. 21, 2009; the contents of all ofthese documents is herein incorporated by reference in their entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Contract No. 5 RO1EB 2991-03, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD

This invention relates to the fields of organic chemistry, polymerchemistry, biochemistry, molecular biology, and medicine. In particularit relates to copolymers (e.g., diblock copolymers) and complexesthereof with polynucleotides to be used as vehicles for delivery of thepolynucleotides into living cells.

BACKGROUND

In certain instances, it is beneficial to provide therapeutic agents(e.g., oligonucleotides) to living cells. In some instances, delivery ofsuch polynucleotides to a living cell provides a therapeutic benefit.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In some embodiments, provided herein are copolymers comprising at leasttwo blocks, the first block comprising at least one constitutional unitthat is hydrophilic (e.g., at about physiologic pH), and the secondblock comprising a plurality of hydrophobic moieties. In certainembodiments, the second block further comprises a chargeable species, ineither the charged or non-charged state, that is anionic at physiologicpH. However, when the pH is at about the pK_(a) of the chargeablespecies, there will exist an equilibrium distribution of chargeablespecies in both forms, that is about 50% will be anionic and about 50%will be non-charged. The further the pH is from the the pK_(a) of thechargeable species, there will be a corresponding shift in thisequilibrium such that at higher pH values, the anionic form willpredominate and at lower pH values, the uncharged form will predominate.The embodiments described herein include the form of the copolymers atany pH value. At a pH value of an endosome (an endosomal pH), thechargeable species will be predominantly in the uncharged form.

Preferably, in certain instances, wherein a polymer described herein isin contact with a cellular membrane, it disrupts or otherwisedestabilizes the membrane and enters the intracellular environment. Inspecific embodiments, a polymer provided herein is endosomolytic orotherwise destabilizing of an endosomal membrane.

In certain embodiments, provided herein is a cellular membranedestabilizing (e.g., an endosomolytic or an endosome-membranedestabilizing) copolymer comprising:

(a) a first block, the first block being a hydrophilic block; and

(b) a second block, the second block being a membrane destabilizinghydrophobic block comprising:

-   -   (i) a first chargeable species that is anionic at about        physiologic pH    -   (ii) a second chargeable species that cationic at about        physiologic pH.

In some embodiments, provided herein is a cellular membranedestabilizing (e.g., an endosomolytic or an endosome-membranedestabilizing) copolymer comprising:

(a) a first block, the first block being a hydrophilic block comprisinga first chargeable species that is cationic at about physiologic pH;

(b) a second block, the second block being a membrane destabilizinghydrophobic block comprising:

-   -   (i) a second chargeable species that is anionic at about neutral        pH; and    -   (ii) a third chargeable species that is cationic at about        physiologic pH; and

(c) an oligonucleotide associated with the first block.

In certain embodiments, provided herein is a cellular membranedestabilizing (e.g., an endosomolytic or an endosome-membranedestabilizing) copolymer comprising:

(a) a first block, the first block being a hydrophilic block;

(b) a second block, the second block being a membrane destabilizinghydrophobic block comprising an acrylic acid residue or alkylacrylicacid residue.

In one aspect the current invention relates to a copolymer (e.g.,diblock copolymer) comprising:

a first block comprising a first constitutional unit that is hydrophilicat normal physiological pH;

a second block comprising:

-   -   a second constitutional unit that is cationic at normal        physiological pH and which can be the same as or different than        the first constitutional unit;    -   a third constitutional unit that is anionic at normal        physiological pH;    -   a hydrophobicity-enhancing moiety wherein:    -   the hydrophobicity-enhancing moiety is covalently bonded to the        second constitutional unit; or,    -   the hydrophobicity enhancing moiety is covalently bonded to the        third constitutional unit; or,        -   the hydrophobicity-enhancing moiety is comprised in a fourth            constitutional unit of the second block; or,        -   any combination of the above; and,    -   the second block is substantially neutral in overall charge.

In various embodiments, the first constitutional unit is cationic atnormal physiological pH (i.e., about physiologic pH), is anionic atnormal physiological pH, is neutral at normal physiological pH, or iszwitterionic at normal physiological pH. In some embodiments, the firstblock of the copolymer is polycationic at normal physiological pH, ispolyanionic at normal physiological pH, is neutral at normalphysiological pH, or is polyzwitterionic at normal physiological pH. Infurther embodiments, the first block of the copolymer has substantiallythe same ionic properties at endosomal pH as at normal physiological pH,e.g., is polycationic at endosomal pH, is polyanionic at endosomal pH,is neutral at endosomal pH, or is polyzwitterionic at endosomal pH.

In one aspect the current invention relates to a copolymer (e.g.,diblock copolymer) comprising:

a first block comprising a first constitutional unit that is cationic atnormal physiological pH;

a second block comprising:

-   -   a second constitutional unit that is cationic at normal        physiological pH and which can be the same as or different than        the first constitutional unit;    -   a third constitutional unit that is anionic at normal        physiological pH;    -   a hydrophobicity-enhancing moiety wherein:        -   the hydrophobicity-enhancing moiety is covalently bonded to            the second constitutional unit; or,        -   the hydrophobicity enhancing moiety is covalently bonded to            the third constitutional unit; or,        -   the hydrophobicity-enhancing moiety is comprised in a fourth            constitutional unit of the second block; or,        -   any combination of the above; and,

the second block is substantially neutral in overall charge.

In an aspect of this invention, the first constitutional unit comprisesa cationic nitrogen species (i.e., a nitrogen species that is cationicat normal physiological pH). In an aspect of this invention the cationicnitrogen species is an ammonium species. In an aspect of this inventionthe second constitutional unit is the same as the first constitutionalunit. In an aspect of this invention the anionic species comprises acarboxylic acid anion. In an aspect of this invention the first blockfurther comprises a charge neutral constitutional unit randomlyinterspersed among the first constitutional units. In an aspect of thisinvention, the first and/or second block comprises at least one reactiveor amenable to modification groups. In an aspect of this invention, ifpresent, the fourth constitutional unit comprises from about 10% toabout 60% by weight of the second block.

In certain embodiments, the first polymer block is approximately 10,000daltons in size, or about 2,000 daltons to about 30,000 daltons, orabout 8,500 daltons to about 13,000 daltons. In some embodiments, thefirst polymer block has a net positive charge similar in absolute valueto the net negative charge on the siRNA molecule being delivered.

In some embodiments, the second polymer block is approximately equal tothe first polymer block in molecular weight, or about 0.2-5 times, orabout 1-3 times the size of the first polymer block and in mostpreferred embodiments the second polymer block is approximately 2-3 (twoto three) times the size of the first polymer block.

In some embodiments, the hydrophilic, charged block is complexed with(used interchangeably herein with “associated with” or “attached to”,e.g., by one or more covalent bond, one or more ionical interaction, acombination thereof, or the like) at least one nucleotide, including apolynucleotide, e.g., an siRNA.

In an aspect of this invention, the polynucleotide block is attached toone of the polymer blocks through an optionally cleavable covalent bond.In an aspect of this invention, the polynucleotide acid is selected fromthe group consisting of DNA, RNA and natural and synthetic analogsthereof. In an aspect of this invention the polynucleotide is antisense.In an aspect of this invention the polynucleotide is RNA. In an aspectof this invention the RNA is selected from the group consisting of mRNA,piRNA, miRNA and siRNA. In an aspect of this invention the RNA is siRNA.

In an aspect of this invention each of the constitutional units isindependently derived from an ethylenic monomer and synthesis of thecopolymer comprises living polymerization.

In an aspect of this invention, the ethylenic monomer is an acrylicmonomer.

Provided in certain embodiments herein is a diblock copolymer, havingthe chemical Formula I:

In some embodiments:

A₀, A₁, A₂, A₃ and A₄ are selected from the group consisting of —C—C—,—C—, —C(O)(C)_(a)C(O)O—, —O(C)_(a)C(O)— and —O(C)_(b)O—; wherein,

-   -   a is 1-4;    -   b is 2-4;

Y₄ is selected from the group consisting of hydrogen, (1C-10C)alkyl,(3C-6C)cycloalkyl, O-(1C-10C)alkyl, —C(O)O(1C-10C)alkyl, C(O)NR₆(1C-10C)and aryl, any of which is optionally substituted with one or morefluorine groups;

Y₀, Y₁ and Y₂ are independently selected from the group consisting of acovalent bond, (1C-10C)alkyl-, —C(O)O(2C-10C) alkyl-, —OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl- and —S(2C-10C)alkyl- —C(O)NR₆(2C-10C) alkyl-;

Y₃ is selected from the group consisting of a covalent bond,(1C-10C)alkyl and (6C-10C)aryl; wherein

-   -   tetravalent carbon atoms of A₁-A₄ that are not fully substituted        with R₁-R₅ and    -   Y₀-Y₄ are completed with an appropriate number of hydrogen        atoms;

each R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from thegroup consisting of hydrogen, —CN, alkyl, alkynyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl and heteroaryl, any of which may beoptionally substituted with one or more fluorine atoms;

Q₀ is a residue selected from the group consisting of residues which arehydrophilic at physiologic pH and are at least partially positivelycharged at physiologic pH (e.g., amino, alkylamino, ammonium,alkylammonium, guanidine, imidazolyl, pyridyl, or the like); at leastpartially negatively charged at physiologic pH but undergo protonationat lower pH (e.g., carboxyl, sulfonamide, boronate, phosphonate,phosphate, or the like); substantially neutral (or non-charged) atphysiologic pH (e.g., hydroxy, polyoxylated alkyl, polyethylene glycol,polypropylene glycol, thiol, or the like); at least partiallyzwitterionic at physiologic pH (e.g., a monomeric residue comprising aphosphate group and an ammonium group at physiologic pH); conjugatableor functionalizable residues (e.g. residues that comprise a reactivegroup, e.g., azide, alkyne, succinimide ester, tetrafluorophenyl ester,pentafluorophenyl ester, p-nitrophenyl ester, pyridyl disulfide, or thelike); or hydrogen;

Q₁ is a residue which is hydrophilic at physiologic pH, and is at leastpartially positively charged at physiologic pH (e.g., amino, alkylamino,ammonium, alkylammonium, guanidine, imidazolyl, pyridyl, or the like);at least partially negatively charged at physiologic pH but undergoesprotonation at lower pH (e.g., carboxyl, sulfonamide, boronate,phosphonate, phosphate, or the like); substantially neutral atphysiologic pH (e.g., hydroxy, polyoxylated alkyl, polyethylene glycol,polypropylene glycol, thiol, or the like); or at least partiallyzwitterionic at physiologic pH (e.g., a monomeric residue comprising aphosphate group and an ammonium group at physiologic pH);

Q₂ is a residue which is positively charged at physiologic pH, includingbut not limited to amino, alkylamino, ammonium, alkylammonium,guanidine, imidazolyl, and pyridyl;

Q₃ is a residue which is negatively charged at physiologic pH, butundergoes protonation at lower pH, including but not limited tocarboxyl, sulfonamide, boronate, phosphonate, and phosphate;

-   -   m is 0 to less than 1.0 (e.g., 0 to about 0.49);    -   n is greater than 0 to 1.0 (e.g., about 0.51 to about 1.0);        wherein

m+n=1

-   -   p is about 0.1 to about 0.9 (e.g., about 0.2 to about 0.5);    -   q is about 0.1 to about 0.9 (e.g., about 0.2 to about 0.5);        wherein:    -   r is 0 to about 0.8 (e.g., 0 to about 0.6); wherein

p+q+r=1

-   -   v is from about 1 to about 25 kDa; and,    -   w is from about 1 to about 50 kDa.

In a specific embodiment, v is about 5 to about 25 kDa. In further oralternative specific embodiments, w is about 1 to about 50 kDa.

In some embodiments, the number or ratio of monomeric residuesrepresented by p and q are within about 30% of each other, about 20% ofeach other, about 10% of each other, or the like. In specificembodiments, p is substantially the same as q. In certain embodiments,at least partially charged generally includes more than a trace amountof charged species, including, e.g., at least 20% of the residues arecharged, at least 30% of the residues are charged, at least 40% of theresidues are charged, at least 50% of the residues are charged, at least60% of the residues are charged, at least 70% of the residues arecharged, or the like.

In certain embodiments, m is 0 and Q₁ is a residue which is hydrophilicand substantially neutral (or non-charged) at physiologic pH. That is,at physiologic pH, any chargeable species on Q₁ is predominantly in aneutral form. In some embodiments, substantially non-charged includes,e.g., less than 5% are charged, less than 3% are charged, less than 1%are charged, or the like. In certain embodiments, m is 0 and Q₁ is aresidue which is hydrophilic and at least partially cationic atphysiologic pH. In certain embodiments, m is 0 and Q₁ is a residue whichis hydrophilic and at least partially anionic at physiologic pH. Incertain embodiments, m is >0 and n is >0 and one of and Q₀ or Q₁ is aresidue which is hydrophilic and at least partially cationic atphysiologic pH and the other of Q₀ or Q₁ is a residue which ishydrophilic and is substantially neutral at physiologic pH. In certainembodiments, m is >0 and n is >0 and one of and Q₀ or Q₁ is a residuewhich is hydrophilic and at least partially anionic at physiologic pHand the other of Q₀ or Q₁ is a residue which is hydrophilic and issubstantially neutral at physiologic pH. In certain embodiments, m is >0and n is >0 and Q₁ is a residue which is hydrophilic and at leastpartially cationic at physiologic pH and Q₀ is a residue which is hconjugatable or functionalizable residues. In certain embodiments, mis >0 and n is >0 and Q₁ is a residue which is hydrophilic andsubstantially neutral at physiologic pH and Q₀ is a residue which is hconjugatable or functionalizable residues.

Provided in certain embodiments herein is copolymer having at least twoblocks, the first block having the chemical Formula Ia, the second blockhaving the chemical Formula Ib, wherein each of the terms describedtherein are as described above:

In certain embodiments, provided herein is a compound of Formula II:

In some embodiments:

A₀, A₁, A₂, A₃ and A₄ are selected from the group consisting of —C—C—,—C(O)(C)_(a)C(O)O—, —O(C)_(a)C(O)— and —O(C)_(b)O—; wherein,

-   -   a is 1-4;    -   b is 2-4;

Y₀ and Y₄ are independently selected from the group consisting ofhydrogen, (1C-10C)alkyl, (3C-6C)cycloalkyl, O-(1C-10C)alkyl,—C(O)O(1C-10C)alkyl, C(O)NR₆(1C-10C) and aryl, any of which isoptionally substituted with one or more fluorine groups;

Y₁ and Y₂ are independently selected from the group consisting of acovalent bond, (1C-10C)alkyl-, —C(O)O(2C-10C) alkyl-, —OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl- and —S(2C-10C)alkyl- —C(O)NR₆(2C-10C) alkyl;

Y₃ is selected from the group consisting of a covalent bond,(1C-10C)alkyl and (6C-10C)aryl; wherein

-   -   tetravalent carbon atoms of A₁-A₄ that are not fully substituted        with R₁-R₅₁ and Y₀-Y₄ are completed with an appropriate number        of hydrogen atoms;

each R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from thegroup consisting of hydrogen, —CN, alkyl, alkynyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl and heteroaryl, any of which may beoptionally substituted with one or more fluorine atoms;

-   -   Q₁ and Q₂ are residues which are positively charged at        physiologic pH, including but not limited to amino, alkylamino,        ammonium, alkylammonium, guanidine, imidazolyl, and pyridyl;    -   Q₃ is a residue which is negatively charged at physiologic pH,        but undergoes protonation at lower pH, including but not limited        to carboxyl, sulfonamide, boronate, phosphonate, and phosphate;        -   m is 0 to about 0.49;        -   n is about 0.51 to about 1.0; wherein

m+n=1

-   -   -   p is about 0.2 to about 0.5;        -   q is about 0.2 to about 0.5; wherein:            -   p is substantially the same as q;        -   r is 0 to about 0.6; wherein

p+q+r=1

-   -   -   v is from about 5 to about 25 kDa; and,        -   w is from about 5 to about 50 kDa.

Provided in some embodiments herein is a diblock copolymer, having (atnormal physiological pH) the chemical formula III:

In some embodiments:

A₀, A₁, A₂, A₃ and A₄ are selected from the group consisting of —C—C—,—C(O)(C)_(a)C(O)O—, —O(C)_(a)C(O)— and —O(C)_(b)O—; wherein,

-   -   a is 1-4;    -   b is 2-4;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independentlyselected from the group consisting of hydrogen, (1C-5C)alkyl,(3C-6C)cycloalkyl and phenyl, any of which may be optionally substitutedwith one or more fluorine atoms;

Y₀ and Y₄ are independently selected from the group consisting ofhydrogen, (1C-10C)alkyl, (3C-6C)cycloalkyl, O-(1C-10C)alkyl,—C(O)O(1C-10C)alkyl and phenyl, any of which is optionally substitutedwith one or more fluorine groups;

Y₁ and Y₂ are independently selected from the group consisting of acovalent bond, (1C-10C)alkyl-, —C(O)O(2C-10C) alkyl-, —OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl- and —S(2C-10C)alkyl-;

Y₃ is selected from the group consisting of a covalent bond,(1C-5C)alkyl and phenyl; wherein

tetravalent carbon atoms of A₁-A₄ that are not fully substituted withR₇-R₁₁ and Y₀-Y₄ are completed with an appropriate number of hydrogenatoms;

Z is a physiologically acceptable counterion,

m is 0 to about 0.49;

n is about 0.51 to about 1.0; wherein

m+n=1

p is about 0.2 to about 0.5;

q is about 0.2 to about 0.5; wherein:

-   -   p is substantially the same as q;

r is 0 to about 0.6; wherein

p+q+r=1

v is from about 5 to about 25 kDa; and,

w is from about 5 to about 50 kDa.

In an aspect of this invention,

A₁ is —C—C—

Y₁ is —C(O)OCH₂CH₂—;

R₆ is hydrogen;

R₇ and R₈ are each —CH₃; and,

R₂ is —CH₃.

In an aspect of this invention,

A₂ is —C—C—;

Y₂ is —C(O)OCH₂CH₂—;

R₉ is hydrogen;

R₁₀ and R₁₁ are each —CH₃; and,

R₃ is —CH₃.

In an aspect of this invention,

A₃ is —C—C—;

R₃ is CH₃CH₂CH₂—;

Y₃ is a covalent bond; and

Z— is a physiologically acceptable anion (e.g., polycation or pluralityof cations).

In certain embodiments:

A₄ is —C—C—;

R₅ is selected from the group consisting of hydrogen and —CH₃; and,

Y₄ is —C(O)O(CH₂)₃CH₃.

In some embodiments:

A₀ is C—C—

R₁ is selected from the group consisting of hydrogen and (1C-3C)alkyl;and

Y₀ is selected from the group consisting of —C(O)O(1C-3C)alkyl.

In some embodiments, m is 0. In certain embodiments, r is 0. In someembodiments, m and r are both 0.

In certain embodiments, provided herein is a method of delivering apolynucleotide into a cell, comprising contacting the cell with apolymer: polynucleotide complex hereof. In specific embodiments, thepolymer: polynucleotide complex is attached in any suitable mannerincluding, by way of non-limiting example, ionic and non-ionicinteractions, such as one or more covalent bond, combinations thereof,or the like. In a specific embodiment, provided herein is a method ofdelivering a polynucleotide into a cell, comprising contacting the cellwith a covalent conjugate of the polymer and polynucleotide.

In an aspect of this invention, the polynucleotide is selected from thegroup consisting of DNA, RNA and natural and synthetic analogs thereof.

In an aspect of this invention, the DNA, RNA or natural or syntheticanalogs thereof is antisense. In an aspect of this invention, thepolynucleotide is RNA. In an aspect of this invention, the RNA is siRNA.In an aspect of this invention, the siRNA is delivered to a cell invivo. In an aspect of this invention, the polymer of this invention isattached or complexed to a targeting moiety. In an aspect of thisinvention, the targeting moiety is covalently attached to the α-end ofthe copolymer (e.g., diblock copolymer). In an aspect of this invention,the targeting moiety is covalently attached to the ω-end of thecopolymer, or is covalently attached to a pendant group of the copolymer(e.g., diblock copolymer). In an aspect of this invention, the targetingmoiety is selected form but not limited to antibodies, antibodyfragments, antibody-like molecules, peptides, cyclic peptides, and smallmolecules.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference for the purposes cited to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A and 1B are illustrative summaries of various polymers describedherein

FIG. 2 is an illustrative synthesis of [PEGMAw]-[B-P-D]

FIGS. 3A, 3B, and 3C are illustrative characterizations ofP7-PEGMA100-40 kDa

FIG. 4 is an illustrative description of the composition and propertiesof PEGMA-DMAEMA copolymers

FIG. 5 is an illustrative synthesis of [PEGMAw-MAA(NHS)]-[B-P-D]

FIGS. 6A, 6B, and 6C are illustrative RAFT copolymerizations of PEGMAand MAA-NHS

FIGS. 7A, 7B, and 7C are illustrative RAFT copolymerizations of DMAEMAand MAA-NHS

FIG. 8 is an illustrative synthesis of PDSMA

FIG. 9 is an illustrative synthesis of HPMA-PDSMA copolymer for siRNAconjugation

FIG. 10A illustrates the hemolysis of polymers and FIG. 10B illustratesthe hemolysis of polymer/siRNA constructs

FIG. 11 illustrates HeLa cell internalization of FAM-labeled siRNA andpolymer/siRNA complexes.

FIG. 12A illustrates nonspecific HeLa cytotoxicity and FIG. 12Billustrates GAPDH knockdown as a function of siRNA polymer carrier

FIGS. 13A and 13B illustrate GAPDH knockdown in HeLas

FIG. 14 illustrates the polymer design forPoly[HPMA]-b-[(PAA)(BMA)(DMAEMA)]

FIG. 15 illustrates the synthesis of pyridyl disulfide-CTA

FIG. 16 illustrates reaction of pyridyl disulfide polymer end group withthe peptide cysteine

FIGS. 17A and 17B illustrate an SDS PAGE gel for characterizingpeptide-polymer conjugates

FIG. 18 illustrates a membrane disruption assay used to measure thecapacity of the polymer to trigger pH-dependent disruption of lipidbilayer membranes

FIGS. 19A and 19B illustrate peptide intracellular localizationfollowing polymer conjugation

FIGS. 20A and 20B illustrate conjugates that lacked the pH-responsiveblock were similar to both control groups and did not result insignificant toxicity

FIGS. 21A and 21B illustrate bioactivity of peptide conjugates.

DETAILED DESCRIPTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Polymer

Provided in certain embodiments, the present invention provides carrierpolymers and polymer: polynucleotide constructs. In certain instances,these polymers and polymer: polynucleotide constructs meet the need fora safe, robust system for delivering therapeutic polynucleotides intocells.

In certain embodiments, provided herein is a cellular membranedestabilizing (e.g., an endosomolytic or an endosome-membranedestabilizing) copolymer comprising:

(a) a first block, the first block being a hydrophilic block; and

(b) a second block, the second block being a membrane destabilizinghydrophobic block comprising:

-   -   (i) a first chargeable species that is anionic at about        physiologic pH    -   (ii) a second chargeable species that cationic at about        physiologic pH.

In some embodiments, provided herein is a cellular membranedestabilizing (e.g., an endosomolytic or an endosome-membranedestabilizing) copolymer comprising:

(a) a first block, the first block being a hydrophilic block comprisinga first chargeable species that is cationic at about physiologic pH;

(b) a second block, the second block being a membrane destabilizinghydrophibic block comprising:

-   -   (i) a second chargeable species that is anionic at about neutral        pH; and    -   (ii) a third chargeable species that is cationic at about        physiologic pH; and

(c) an oligonucleotide associated with the first block.

In specific embodiments of the polymers described herein, eachchargeable species is present on a different constitutional unit. Insome embodiments, a first constitutional unit comprises the firstchargeable species. In further or alternative embodiments, a secondconstitutional unit comprises the second chargeable species. In furtheror alternative embodiments, a third constitutional unit comprises thethird chargeable species.

Some of the constitutional units of this invention are stated to becationic or anionic at normal physiological pH. Thus, in certaininstances, at normal physiological pH, the species have a pKa thatresults in it being protonated (cationic, positively charged) ordeprotonated (anionic, negatively charged). Presently preferred cationicspecies at physiological pH are nitrogen species such as ammonium,—NRR′R″, guanidinium (—NRC(═NR′H)+NR″R′″, ignoring canonical forms thatare known to those skilled in the art) wherein the R groups areindependently hydrogen, alkyl, cycloalkyl or aryl or two R groups bondedto the same or adjacent nitrogen atoms may be also be joined to oneanother to form a heterocyclic species such as pyrrole, imidazole,indole and the like. Monomeric residues or constitutional unitsdescribed herein as cationic at normal physiological pH comprise aspecies charged or chargeable to a cation, including a deprotonatablecationic species.

In various embodiments described herein, constitutional units, that arecationic or positively charged at physiological pH (including, e.g.,certain hydrophilic constitutional units) described herein comprise oneor more amino groups, alkylamino groups, guanidine groups, imidazolylgroups, pyridyl groups, or the like, or the protonated, alkylated orotherwise charged forms thereof. In some embodiments, constitutionalunits that are cationic at normal physiological pH that are utilizedherein include, by way of non-limiting example, monomeric residues ofdialkylaminoalkylmethacrylates (e.g., DMAEMA). In various embodimentsdescribed herein, constitutional units, that are anionic or negativelycharged at physiological pH (including, e.g., certain hydrophilicconstitutional units) described herein comprise one or more acid groupor conjugate base thereof, including, by way of non-limiting example,carboxylate, sulfonamide, boronate, phosphonate, phosphate, or the like.In some embodiments, constitutional units that are anionic or negativelycharged at normal physiological pH that are utilized herein include, byway of non-limiting example, monomeric residues of acrylic acid, alkylacrylic acid (e.g., methyl acrylic acid, ethyl acrylic acid, propylacrylic acid, etc.), or the like. In various embodiments describedherein, hydrophilic constitutional units that are neutral at physiologicpH comprise one or more hydrophilic group, e.g., hydroxy, polyoxylatedalkyl, polyethylene glycol, polypropylene glycol, thiol, or the like. Insome embodiments, hydrophilic constitutional units that are neutral atnormal physiological pH that are utilized herein include, by way ofnon-limiting example, monomeric residues of PEGylated acrylic acid,PEGylated methacrylic acid, hydroxyalkylacrylic acid,hydroxyalkylalkacrylic acid (e.g, HPMA), or the like. In variousembodiments described herein, hydrophilic constitutional units that arezwitterionic at physiologic pH comprise an anionic or negatively chargedgroup at physiologic pH and a cationic or positively charged group atphysiologic pH. In some embodiments, hydrophilic constitutional unitsthat are zwitterionic at normal physiological pH that are utilizedherein include, by way of non-limiting example, monomeric residues ofcomprising a phosphate group and an ammonium group at physiologic pH,such as set forth in U.S. Pat. No. 7,300,990, which is herebyincorporated herein for such disclosure, or the like.

In certain embodiments, polymers provided herein further comprise one ormore constitutional unit comprising a conjugatable or functionalizableside chain (e.g., a pendant group of a monomeric residue). In someinstances, a conjugatable or functionalizable side chain is a groupbearing one or more reactive groups that can be used forpost-polymerization introduction of additional functionalities via knowin the art chemistries, for example, “click” chemistry (for example of“click” reactions, see Wu, P.; Fokin, V. V. Catalytic Azide-AlkyneCycloaddition: Reactivity and Applications. Aldrichim. Acta, 2007, 40,7-17). In certain embodiments, conjugatable or functionalizable sidechains provided herein comprise one or more of any suitable activatedgroup, such as but not limited to N-hydrosuccinimide (NHS)ester, HOBt(1-hydroxybenzotriazole) ester, p-nitrophenyl ester, tetrafluorophenylester, pentafluorophenyl ester, pyridyl disulfide group or the like.

In some embodiments, constitutional units that are anionic at normalphysiological pH comprise carboxylic acids such as, without limitation,monomeric residues of 2-propyl acrylic acid (i.e., the constitutionalunit derived from it, 2-propylpropionic acid, —CH₂C((CH₂)₂CH₃)(COOH)—(PAA)), although any organic or inorganic acid that can be present,either as a protected species, e.g., an ester, or as the free acid, inthe selected polymerization process is also within the contemplation ofthis invention. Anionic monomeric residues or constitutional unitsdescribed herein comprise a species charged or chargeable to an anion,including a protonatable anionic species. In certain instances, anionicmonomeric residues can be anionic at neutral pH 7.0.

Monomers such as maleic-anhydride, (Scott M. Henry, Mohamed E. H.El-Sayed, Christopher M. Pirie, Allan S. Hoffman, and Patrick S. Stayton“pH-Responsive Poly(styrene-alt-maleic anhydride) Alkylamide Copolymersfor Intracellular Drug Delivery” Biomacromolecules 7:2407-2414, 2006)may also be used for introduction of negatively charged units (e.g., thethird constitutional unit) into the second block. In such embodiments,the negatively charged constitutional unit is a maleic anhydridemonomeric residue.

An embodiment of this invention is a polymer having the followinggeneral structure of Formula I:

In some embodiments:

A₀, A₁, A₂, A₃ and A₄ are selected from the group consisting of —C—,—C—C—, —C(O)(C)_(a)C(O)O—, —O(C)_(a)C(O)— and —O(C)_(b)O—; wherein,

-   -   a is 1-4;    -   b is 2-4;

Y₄ is selected from the group consisting of hydrogen, (1C-10C)alkyl,(3C-6C)cycloalkyl, O-(1C-10C)alkyl, —C(O)O(1C-10C)alkyl,C(O)NR₆(1C-10C), (4C-10C)heteroaryl and (6C-10C)aryl, any of which isoptionally substituted with one or more fluorine groups;

Y₀, Y₁ and Y₂ are independently selected from the group consisting of acovalent bond, (1C-10C)alkyl-, —C(O)O(2C-10C) alkyl-, —OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl- and —S(2C-10C)alkyl-, —C(O)NR₆(2C-10C) alkyl-,-(4C-10C)heteroaryl- and -(6C-10C)aryl-;

Y₃ is selected from the group consisting of a covalent bond,-(1C-10C)alkyl-, -(4C-10C)heteroaryl- and -(6C-10C)aryl-; wherein

-   -   tetravalent carbon atoms of A₁-A₄ that are not fully substituted        with R₁-R₅ and Y₀—Y₄ are completed with an appropriate number of        hydrogen atoms;

R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from the groupconsisting of hydrogen, —CN, alkyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl, any of which may be optionallysubstituted with one or more fluorine atoms;

Q₀ is a residue selected from the group consisting of residues which arehydrophilic at physiologic pH, and are at least partially positivelycharged at physiologic pH (e.g., amino, alkylamino, ammonium,alkylammonium, guanidine, imidazolyl, pyridyl, or the like); at leastpartially negatively charged at physiologic pH but undergo protonationat lower pH (e.g., carboxyl, sulfonamide, boronate, phosphonate,phosphate, or the like); substantially neutral (or non-charged) atphysiologic pH (e.g., hydroxy, polyoxylated alkyl, polyethylene glycol,polypropylene glycol, thiol, or the like); at least partiallyzwitterionic at physiologic pH (e.g., a monomeric residue comprising aphosphate group and an ammonium group at physiologic pH); conjugatableor functionalizable residues (e.g. residues that comprise a reactivegroup, e.g., azide, alkyne, succinimide ester, tetrafluorophenyl ester,pentafluorophenyl ester, p-nitrophenyl ester, pyridyl disulfide, or thelike); or hydrogen;

Q₁ is a residue which is hydrophilic at physiologic pH, and is at leastpartially positively charged at physiologic pH (e.g., amino, alkylamino,ammonium, alkylammonium, guanidine, imidazolyl, pyridyl, or the like);at least partially negatively charged at physiologic pH but undergoesprotonation at lower pH (e.g., carboxyl, sulfonamide, boronate,phosphonate, phosphate, or the like); substantially neutral atphysiologic pH (e.g., hydroxy, polyoxylated alkyl, polyethylene glycol,polypropylene glycol, thiol, or the like); or at least partiallyzwitterionic at physiologic pH (e.g., comprising a phosphate group andan ammonium group at physiologic pH);

Q₂ is a residue which is positively charged at physiologic pH, includingbut not limited to amino, alkylamino, ammonium, alkylammonium,guanidine, imidazolyl, and pyridyl;

Q₃ is a residue which is negatively charged at physiologic pH, butundergoes protonation at lower pH, including but not limited tocarboxyl, sulfonamide, boronate, phosphonate, and phosphate;

-   -   m is about 0 to less than 1.0 (e.g., 0 to about 0.49);    -   n is greater than 0 to about 1.0 (e.g., about 0.51 to about        1.0); wherein

m+n=1

-   -   p is about 0.1 to about 0.9 (e.g., about 0.2 to about 0.5);    -   q is about 0.1 to about 0.9 (e.g., about 0.2 to about 0.5);        wherein:    -   r is 0 to about 0.8 (e.g., 0 to about 0.6); wherein

p+q+r=1

-   -   v is from about 1 to about 25 kDa, or about 5 to about 25 kDa;        and,    -   w is from about 1 to about 50 kDa, or about 5 to about 50 kDa.

In some embodiments, the number or ratio of monomeric residuesrepresented by p and q are within about 30% of each other, about 20% ofeach other, about 10% of each other, or the like. In specificembodiments, p is substantially the same as q. In certain embodiments,at least partially charged generally includes more than a trace amountof charged species, including, e.g., at least 20% of the residues arecharged, at least 30% of the residues are charged, at least 40% of theresidues are charged, at least 50% of the residues are charged, at least60% of the residues are charged, at least 70% of the residues arecharged, or the like.

In certain embodiments, m is 0 and Q₁ is a residue which is hydrophilicand substantially neutral (or non-charged) at physiologic pH. In someembodiments, substantially non-charged includes, e.g., less than 5% arecharged, less than 3% are charged, less than 1% are charged, or thelike. In certain embodiments, m is 0 and Q₁ is a residue which ishydrophilic and at least partially cationic at physiologic pH. Incertain embodiments, m is 0 and Q₁ is a residue which is hydrophilic andat least partially anionic at physiologic pH. In certain embodiments, mis >0 and n is >0 and one of and Q₀ or Q₁ is a residue which ishydrophilic and at least partially cationic at physiologic pH and theother of Q₀ or Q₁ is a residue which is hydrophilic and is substantiallyneutral at physiologic pH. In certain embodiments, m is >0 and n is >0and one of and Q₀ or Q₁ is a residue which is hydrophilic and at leastpartially anionic at physiologic pH and the other of Q₀ or Q₁ is aresidue which is hydrophilic and is substantially neutral at physiologicpH. In certain embodiments, m is >0 and n is >0 and Q₁ is a residuewhich is hydrophilic and at least partially cationic at physiologic pHand Q₀ is a residue which is a conjugatable or functionalizable residue.In certain embodiments, m is >0 and n is >0 and Q₁ is a residue which ishydrophilic and substantially neutral at physiologic pH and Q₀ is aresidue which is a conjugatable or functionalizable residue.

In some embodiments, the positively charged or at least partiallypositively charged at physiologic pH group is a —NR′R″ group, wherein R′and R″ are independently selected from hydrogen, alkyl, cycloalkyl, orheteroalkyl which may be optionally substituted with one or morehalogen, amino, hydroxyl groups and/or comprise one or more unsaturatedbonds; in some embodiments, R′ and R′ are taken together to form asubstituted of unsubstituted heteroaryl or alicyclic heterocycle. Insome embodiments, groups described herein as positively charged or atleast partially positively charged at physiologic pH may include, by wayof non-limiting example, amino, alkyl amino, dialkyl amino, cyclic amino(e.g., piperidine or N-alkylated piperidine), alicyclic imino (e.g.,dihydro-pyridinyl, 2,3,4,5-tetrahydro-pyridinyl, or the like),heteroaryl imino (e.g., pyridinyl), or the like. In some embodiments,groups described herein as negatively charged or at least partiallynegatively charged at physiologic pH but undergoes protonation at lowerpH, such as, by way of non-limiting example, carboxylic acid (COOH),sulfonamide, boronic acid, sulfonic acid, sulfinic acid, sulfuric acid,phosphoric acid, phosphinic acid, phosphorous acid, carbonic acid, thedeprotonated conjugate base thereof, or the like.

In certain embodiments, provided herein is a compound of Formula II:

In some embodiments:

A₀, A₁, A₂, A₃ and A₄ are selected from the group consisting of —C—C—,—C(O)(C)_(a)C(O)O—,

—O(C)_(a)C(O)— and —O(C)_(b)O—; wherein,

-   -   a is 1-4;    -   b is 2-4;

Y₀ and Y₄ are independently selected from the group consisting ofhydrogen, (1C-10C)alkyl, (3C-6C)cycloalkyl, O-(1C-10C)alkyl,—C(O)O(1C-10C)alkyl, C(O)NR₆(1C-10C) and aryl, any of which isoptionally substituted with one or more fluorine groups;

Y₁ and Y₂ are independently selected from the group consisting of acovalent bond, (1C-10C)alkyl-, —C(O)O(2C-10C) alkyl-, —OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl- and —S(2C-10C)alkyl- —C(O)NR₆(2C-10C) alkyl;

Y₃ is selected from the group consisting of a covalent bond,(1C-10C)alkyl and (6C-10C)aryl; wherein

-   -   tetravalent carbon atoms of A1-A4 that are not fully substituted        with R₁-R₅ and    -   Y₀-Y₄ are completed with an appropriate number of hydrogen        atoms;

R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from the groupconsisting of hydrogen, —CN, alkyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl, any of which may be optionallysubstituted with one or more fluorine atoms;

Q₁ and Q₂ are residues which are positively charged at physiologic pH,including but not limited to amino, alkylamino, ammonium, alkylammonium,guanidine, imidazolyl, and pyridyl.

Q₃ is a residue which is negatively charged at physiologic pH, butundergoes protonation at lower pH, including but not limited tocarboxyl, sulfonamide, boronate, phosphonate, and phosphate.

m is 0 to about 0.49;

n is about 0.51 to about 1.0; wherein

m+n=1

-   -   p is about 0.2 to about 0.5;    -   q is about 0.2 to about 0.5; wherein:        -   p is substantially the same as q;

r is 0 to about 0.6; wherein

p+q+r=1

-   -   v is from about 1 to about 25 kDa, or about 5 to about 25 kDa;        and,    -   w is from about 1 to about 50 kDa, or about 5 to about 50 kDa.

In certain embodiments, the block copolymer is a diblock copolymer,having the chemical formula (at normal physiological or about neutralpH) of Formula III:

In certain embodiments, A₀, A₁, A₂, A₃, and A₄, substituted as indicatedcomprise the constitutional units (used interchangeably herein with“monomeric units” and “monomeric residues”) of the polymer of FormulaIII. In specific embodiments, the monomeric units of constituting the Agroups of Formula III are polymerizably compatible under appropriateconditions. In certain instances, an ethylenic backbone orconstitutional unit, —(C—C—)_(m)— polymer, wherein each C isdi-substituted with H and/or any other suitable group, is polymerizedusing monomers containing a carbon-carbon double bond, >C═C<. In certainembodiments, each A group (e.g., each of A₀, A₁, A₂, A₃, and A₄) may be(i.e., independently selected from) —C—C— (i.e., an ethylenic monomericunit or polymer backbone), —C(O)(C)_(a)C(O)O— (i.e., a polyanhydridemonomeric unit or polymer backbone), —O(C)_(a)C(O)— (i.e., a polyestermonomeric unit or polymer backbone), —O(C)_(b)O— (i.e., a polyalkyleneglycol monomeric unit or polymer backbone), or the like (wherein each Cis di-substituted with H and/or any other suitable group such asdescribed herein, including R₁₂ and/or R₁₃ as described above). Inspecific embodiments, the term “a” is an integer from 1 to 4, and “b” isan integer from 2 to 4. In certain instances, each “Y” and “R” groupattached to the backbone of Formula III (i.e., any one of Y₀, Y₁, Y₂,Y₃, Y₄, R₁, R₂, R₃, R₄, R₅) is bonded to any “C” (including any (C)_(a)or (C)_(b)) of the specific monomeric unit. In specific embodiments,both the Y and R of a specific monomeric unit is attached to the same“C”. In certain specific embodiments, both the Y and R of a specificmonomeric unit is attached to the same “C”, the “C” being alpha to thecarbonyl group of the monomeric unit, if present.

In specific embodiments, R₁-R₁₁ are independently selected fromhydrogen, alkyl (e.g., 1C-5C alkyl), cycloalkyl (e.g., 3C-6Ccycloalkyl), or phenyl, wherein any of R₁-R₁₁ is optionally substitutedwith one or more fluorine, cycloalkyl, or phenyl, which may optionallybe further substituted with one or more alkyl group.

In certain specific embodiments, Y₀ and Y₄ are independently selectedfrom hydrogen, alkyl (e.g., 1C-10C alkyl), cycloalkyl (e.g., 3C-6Ccycloalkyl), O-alkyl (e.g., 0-(2C-10C)alkyl, —C(O)O-alkyl (e.g.,—C(O)O-(2C-10C)alkyl), or phenyl, any of which is optionally substitutedwith one or more fluorine.

In some embodiments, Y₁ and Y₂ are independently selected from acovalent bond, alkyl, preferably at present a (1C-10C)alkyl,—C(O)O-alkyl, preferably at present —C(O)O-(2C-10C)alkyl,

—OC(O)alkyl, preferably at present —OC(O)-(2C-10C)alkyl, O-alkyl,preferably at present —O(2C-10C)alkyl and —S-alkyl, preferably atpresent —S-(2C-10C)alkyl. In certain embodiments, Y₃ is selected from acovalent bond, alkyl, preferably at present (1C-5C)alkyl and phenyl.

In some embodiments, Z— is present or absent. In certain embodiments,wherein R₁ and/or R₄ is hydrogen, Z— is OH—. In certain embodiments, Z—is any counterion (e.g., one or more counterion), preferably abiocompatible counter ion, such as, by way of non-limiting example,chloride, inorganic or organic phosphate, sulfate, sulfonate, acetate,propionate, butyrate, valerate, caproate, caprylate, caprate, laurate,myristate, palmate, stearate, palmitolate, oleate, linolate, arachidate,gadoleate, vaccinate, lactate, glycolate, salicylate,desamionphenylalanine, desaminoserine, desaminothreonine,ε-hydroxycaproate, 3-hydroxybutylrate, 4-hydroxybutyrate or3-hydroxyvalerate. In some embodiments, when each Y, R and optionalfluorine is covalently bonded to a carbon of the selected backbone, anycarbons that are not fully substituted are completed with theappropriate number of hydrogen atoms. The numbers m, n, p, q and rrepresent the mole fraction of each constitutional unit in its block andv and w provide the molecular weight of each block.

In certain embodiments,

A₀, A₁, A₂, A₃ and A₄ are selected from the group consisting of —C—,—C—C—, —C(O)(CR₁₂R₁₃)_(a)C(O)O—, —O(CR₁₃R₁₃)_(a)C(O)— andO(CR₁₃R₁₃)_(b)O; wherein,

-   -   a is 1-4;    -   b is 2-4;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, and R₁₃ areindependently selected from the group consisting of hydrogen,(1C-5C)alkyl, (3C-6C)cycloalkyl, (5C-10C)aryl, (4C-10C)heteroaryl, anyof which may be optionally substituted with one or more fluorine atoms;

Y₀ and Y₄ are independently selected from the group consisting ofhydrogen, (1C-10C)alkyl, (3C-6C)cycloalkyl, O-(1C-10C)alkyl,—C(O)O(1C-10C)alkyl and phenyl, any of which is optionally substitutedwith one or more fluorine groups;

Y₁ and Y₂ are independently selected from the group consisting of acovalent bond, (1C-10C)alkyl-, —C(O)O(2C-10C) alkyl-, —OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl- and —S(2C-10C)alkyl-;

Y₃ is selected from the group consisting of a covalent bond,(1C-5C)alkyl and phenyl; wherein tetravalent carbon atoms of A1-A4 thatare not fully substituted with R₁-R₅ and Y₀-Y₄ are completed with anappropriate number of hydrogen atoms;

Z is one or more physiologically acceptable counterions,

m is 0 to about 0.49;

n is about 0.51 to about 1.0; wherein

m+n=1

p is about 0.2 to about 0.5;

q is about 0.2 to about 0.5; wherein:

-   -   p is substantially the same as q;

r is 0 to about 0.6; wherein

p+q+r=1

v is from about 1 to about 25 kDa, or about 5 to about 25 kDa; and,

w is from about 1 to about 50 kDa, or about 5 to about 50 kDa.

In a specific embodiment,

A₀, A₁, A₂, A₃ and A₄ are independently selected from the groupconsisting of —C—C—, —C(O)(C)_(a)C(O)O—, —O(C)_(a)C(O)— and —O(C)_(b)O—;wherein,

-   -   a is 1-4;    -   b is 2-4;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and R₁₁ are independentlyselected from the group consisting of hydrogen, (1C-5C)alkyl,(3C-6C)cycloalkyl and phenyl, any of which may be optionally substitutedwith one or more fluorine atoms;

Y₀ and Y₄ are independently selected from the group consisting ofhydrogen, (1C-10C)alkyl, (3C-6C)cycloalkyl, O-(1C-10C)alkyl,—C(O)O(1C-10C)alkyl and phenyl, any of which is optionally substitutedwith one or more fluorine groups;

Y₁ and Y₂ are independently selected from the group consisting of acovalent bond, (1C-10C)alkyl-, —C(O)O(2C-10C) alkyl-, —OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl- and —S(2C-10C)alkyl-;

Y₃ is selected from the group consisting of a covalent bond,(1C-5C)alkyl and phenyl;

wherein tetravalent carbon atoms of A₁-A₄ that are not fully substitutedwith R₁-R₅ and Y₀—Y₄ are completed with an appropriate number ofhydrogen atoms;

Z is a physiologically acceptable counterion,

m is 0 to about 0.49;

n is about 0.51 to about 1.0;

wherein m+n=1

p is about 0.2 to about 0.5;

q is about 0.2 to about 0.5; wherein:

-   -   p is substantially the same as q;

r is 0 to about 0.6; wherein

p+q+r=1

-   -   v is from about 5 to about 25 kDa; and    -   w is from about 5 to about 25 kDa.

In some embodiments,

A₁ is —C—C—

Y₁ is —C(O)OCH₂CH₂—;

R₆ is hydrogen;

R₇ and R₈ are each —CH₃; and,

R₂ is —CH₃.

In some embodiments,

A₂ is —C—C—;

Y₂ is —C(O)OCH₂CH₂—;

R₉ is hydrogen;

R₁₀ and R₁₁ are each —CH₃; and,

R₃ is —CH₃.

In some embodiments,

A₃ is —C—C—;

R₄ is CH₃CH₂CH₂—;

Y₃ is a covalent bond;

and Z— is a physiologically acceptable anion.

In some embodiments,

A₄ is —C—C—;

R₅ is selected from the group consisting of hydrogen and —CH₃; and,

Y₄ is —C(O)O(CH₂)₃CH₃.

In some embodiments,

A₀ is C—C—

R₁ is selected from the group consisting of hydrogen and (1C-3C)alkyl;and,

Y₀ is selected from the group consisting of —C(O)O(1C-3C)alkyl.

In some embodiments, m is 0.

In some embodiments, r is 0.

In some embodiments, m and r are both 0.

Provided in some embodiments, is an exemplary but non-limiting polymerof this invention:

In certain instances, the constitutional units of compound I are asshown within the square bracket on the left and the curved brackets onthe right and they are derived from the monomers:

The letters p, q and r represent the mole fraction of eachconstitutional unit within its block. The letters v and w represent themolecular weight (number average) of each block in the diblockcopolymer.

Provided in some embodiments, a compound provided herein is a compoundhaving the structure:

As discussed above, letters p, q and r represent the mole fraction ofeach constitutional unit within its block. The letters v and w representthe molecular weight (number average) of each block in the diblockcopolymer.

In some embodiments, provided herein the following polymers:

[DMAEMA]_(v)-[B_(p)-/-P_(q)-/-D_(r)]_(w)  IV3

[PEGMA]_(v)-[B_(p)-/-P_(q)-/-D_(r)]_(w)  IV4

[PEGMA_(m)-/-DMAEMA_(n)]_(v)-[B_(p)-/-P_(q)-/-D_(r)]_(w)  IV5

[PEGMA_(m)-/-MAA(NHS)_(n)]d_(v)-[B_(p)-/-P_(q)-/-D_(r)]_(w)  IV6

[DMAEMA_(m)-/-MAA(NHS)_(n)]_(v)-[B_(p)-/-P_(q)-/-D_(r)]_(w)  IV7

[HPMA_(m)-/-PDSM_(n)]_(v)-[B_(p)-/-P_(q)-/-D_(r)]_(w)  IV8

[PEGMA_(m)-/-PDSM_(n)]_(v)-[B_(p)-/-P_(q)-/-D_(r)]_(w)  IV9

In some embodiments, B is butyl methacrylate residue; P is propylacrylic acid residue; D and DMAEMA are dimethylaminoethyl methacrylateresidue; PEGMA is polyethyleneglycol methacrylate residue (e.g., with1-20 ethylene oxide units, such as illustrated in compound IV-2, or 4-5ethylene oxide units, or 7-8 ethylene oxide units); MAA(NHS) ismethylacrylic acid-N-hydroxy succinimide residue; HPMA isN-(2-hydroxypropyl) methacrylamide residue; and PDSM is pyridyldisulfide methacrylate residue. In certain embodiments, the terms m, n,p, q, r, w and v are as described herein. In specific embodiments, w isabout 0.1 times to about 5 times v, or about 1 times to about 5 times v.

Polymers IV1-IV9 are examples of polymers provided herein comprising avariety of constitutional unit(s) making up the first block of thepolymer. Moreover, polymers set forth in the Figures and Table, as wellas structurally related polymers (such as variations in MW and/ormonomeric residue ratios) are specifically provided for herein. In someembodiments, the constitutional unit(s) of the first block are varied orchemically treated in order to create polymers where the first block isor comprises a constitutional unit that is neutral (e.g., PEGMA),cationic (e.g., DMAEMA), anionic (e.g., PEGMA-NHS, where the NHS ishydrolyzed to the acid, or acrylic acid), ampholytic (e.g., DMAEMA-NHS,where the NHS is hydrolyzed to the acid), or zwiterrionic (for example,poly[2-methacryloyloxy-2′trimethylammoniumethyl phosphate]). In someembodiments, polymers comprising pyridyl disulfide functionality in thefirst block, e.g., [PEGMA PDSM]-[B-P-D], that can be and is optionallyreacted with a thiolated siRNA to form a polymer-siRNA conjugate.

In some embodiments, the polymers of this invention are “diblockcopolymers.” The term “copolymer” signifies that the polymer is theresult of polymerization of two or more different monomers. In someinstances, a “block” copolymer refers to a structure in which distinctsub combinations of constitutional units are joined together. In certaininstances, a “block” refers to a segment or portion of a polymer havinga particular characteristics (e.g., a hydrophilic segment or ahydrophobic segment of a gradient copolymer). In some instances, adiblock copolymer comprises just two blocks; a schematic generalizationof such a polymer would look like: [A_(a)B_(b)C_(c) . . .]_(m)-[X_(x)Y_(y)Z_(z) . . . ]_(n) wherein each letter stands for aconstitutional unit, each subscript to a constitutional unit representsthe mole fraction of that unit in the particular block, the three dotsindicate that there may be more (but of course there may also be fewer)constitutional units in each block and m and n indicate the molecularweight of each block in the diblock copolymer. As suggested by theschematic, the number and the nature of each constitutional unit isseparately controlled for each block. It is understood that theschematic is not meant and should not be construed to infer anyrelationship whatsoever between the number of constitutional units orthe number of different types of constitutional units in each of theblocks. Nor is the schematic meant to describe any particulararrangement of the constitutional units within a particular block. Thatis, in each block the constitutional units may be disposed in a purelyrandom, an alternating random, a regular alternating, a regular block ora random block configuration unless expressly stated to be otherwise. Apurely random configuration would, for example, be:x-x-y-z-x-y-y-z-y-z-z-z . . . or y-z-x-y-z-y-z-x-x . . . . Analternating random configuration would be: x-y-x-z-y-x-y-z-y-x-z . . . ,and a regular alternating configuration would be: x-y-z-x-y-z-x-y-z . .. . A regular block configuration has the following generalconfiguration: . . . x-x-x-y-y-y-z-z-z-x-x-x . . . , while a randomblock configuration has the general configuration: . . .x-x-x-z-z-x-x-y-y-y-y-z-z-z-x-x-z-z-z- . . . . In none of the precedinggeneric examples is the particular juxtaposition of individualconstitutional units or blocks or the number of constitutional units ina block or the number of blocks meant nor should they be construed as inany manner bearing on or limiting the actual structure of diblockcopolymers of this invention.

It is further understood that the curved brackets enclosing theconstitutional units are not meant and are not to be construed to meanthat the constitutional units themselves form blocks. That is, theconstitutional units within the square brackets may combine in anymanner with the other constitutional units within the block, i.e.,purely random, alternating random, regular alternating, regular block orrandom block configurations. Thus in diblock copolymer l, p, q and rrepresent the mole fraction of that constitutional unit in the block andis not intended, and must not be construed, as indicating or suggestingthat the constitutional units within the brackets comprise a blockwithin a block.

Thus, when it is stated herein that a charge neutral constitutional unitmay be “randomly interspersed” among the first constitutional units ofthe first block of a diblock copolymer of this invention, it means thatthe first block would have a structure generically akin to thatdescribed above for a purely random configuration.

In some embodiments, the solubility of any of the block copolymersdescribed herein in aqueous solution or medium at about neutral pH ismore than 1 mg/mL, more than 5 mg/mL, more than 10 mg/mL, more than 25mg/mL, more than 50 mg/mL, more than 100 mg/mL and more than 500 mg/mL.In some embodiments, in particular for diblock polymers having ahydrophilic (e.g., a cationic hydrophilic) first block, the threespecies present in the hydrophobic block (anionic, cationic andhydrophobic) are present as a random copolymer block, or are otherwisepresent in an interspersed sequence such that the block is ofapproximately net neutral charge across its length. In some instances,this orientation provides increased solubility of the block copolymer.

In certain embodiments, the diblock copolymers set forth in Table 1 areprovided herein; in certain instances, such polymers are used aspolynucleotide complexing agents and carriers. It is understood that thecharacteristics of diblock copolymers described in Table 1 and otherwiseherein will be translatable to other diblock copolymers hereof suchthat, based on the disclosures herein, those skilled in the art will becapable of preparing such copolymers, which will therefore be within thescope of this invention.

The first block of exemplary diblock copolymers are composed ofmonomeric residues of dimethylaminoethylmethacrylate (DMAEMA), whichefficiently binds to and condenses nucleic acids at physiological pH.The second block of an exemplary polymer described herein containedmonomeric residues of DMAEMA as a cationic constitutional unit;monomeric residues of propyl acrylic acid (PAA) as an anionicconstitutional unit and, due to the hydrophobic propyl substituent, acontributor to the hydrophobicity enhancing moiety; and monomericresidues of butyl methylacrylate (BMA) as a separate constitutionalunit, constituting or comprising a hydrophobicity enhancing moiety. Incertain instances, the second block enables endosomal escape of thebound nucleic acid through a pH-induced conformational change which, insome instances, results in membrane destabilization. In some instances,under physiological conditions, the second or hydrophobic core block hasboth positive (e.g., protonated DMAEMA) residues and negative (e.g.,de-protonated PAA) residues in similar amounts, resulting in approximatecharge neutrality and charge stabilization of the core by the formationof ion pairs. In certain instances, upon uptake of a polymer nucleicacid composition described herein into endosomal compartments of thecell, the lower pH of the endosomal environment causes anionic residuesof the third constitutional unit (e.g., PAA carboxylate groups) tobecome protonated and thereby membrane disruptive. In some instances,protonation or neutralization some or all of the anionic residuesresults in charge neutralization of the PAA acidic residues and, incertain instances, in a conformational change in the polymer to ahydrophobic membrane-destabilizing form.

TABLE 1 Molecular weights, polydispersities, and monomer compositionsfor the poly(DMAEMA) macroCTA, the resultant diblock copolymers andtheir corresponding nomenclature. M_(n) ^(a) M_(n) ^(a) TheoreticalTheoretical Theoretical Experimental^(b) Experimental^(b)Experimental^(b) 1st block 2nd block % BMA % PAA % DMAEMA % BMA % PAA %DMAEMA Polymer (g/mol) (g/mol) PDI^(a) 2^(nd) block 2^(nd) block 2^(nd)block 2^(nd) block 2^(nd) block 2^(nd) block mCTA 9 100 — 1.16 — — — — —— P1 9 100 6 900 1.58 0 50 50 — 47 53 P2 9 100 8 900 1.56 5 47.5 47.5 148 51 P3 9 100 8 300 1.54 10 45 45 12 40 48 P4 9 100 9 300 1.46 15 42.542.5 19 44 37 P5 9 100 10 100  1.51 20 40 40 24 40 36 P6 9 100 10 000 1.48 30 35 35 27 37 36 P7 9 100 11 300  1.45 40 30 30 48 29 23 ^(a)Asdetermined by SEC Tosoh TSK-GEL R-3000 and R-4000 columns (TosohBioscience, Mongomeryville, PA) connected in series to a Viscotek GPCmaxVE2001 and refractometer VE3580 (Viscotek, Houston, TX). HPLC-grade DMFcontaining 0.1 wt % LiBr was used as the mobile phase. The molecularweights of the synthesized copolymers were determined using series ofpoly(methyl methacrylate) standards. ^(b)As determined by ¹H NMRspectroscopy (3 wt % in CDCL₃; Bruker DRX 499)

Poly(DMAEMA) and other polymeric entities used herein (e.g., copolymersor copolymer blocks of BMA, DMAEMA and PAA) are prepared in any suitablemanner. In one instance, poly(DMAEMA) was prepared by polymerizingDMAEMA in the presence of the RAFT CTA, ECT, and a radical initiator. Insome instances, a block, poly(DMAEMA) (9,100 g/mol; DP 58), was used toprepare a series of diblock copolymers where the BMA content wasincreased and equimolar quantities of DMAEMA and PAA were maintained.Characteristics of the resulting polymers are shown in Table 1. Similarblock sizes were observed for all seven diblocks giving the polymers anoverall molecular weight of around 20,000 g/mol. In certain embodiments,lower molecular weights are chosen. In some instances, lower molecularweight polymers minimize polymer toxicity and enable renal clearance ofthe polymers in order to ensure amenable translation to in vivo testing.Also shown in Table 1 are the theoretical and experimentally derivedmonomer compositions of the second block. Each polymer listed is anexample of a class of related polymers. For example, polymers of the P7class have several versions, one of which is characterized in Table 1.While all polymers are relatively close to the theoretical composition,some deviation is observed in all cases and is likely due to differencesin the monomer reactivity ratios.

Alternatively, the orientation of the blocks on the diblock polymer isreversed, such that the ω-end of the polymer is the hydrophilic block.In various embodiments, this is achieved in any suitable manner,including a number of ways synthetically. For example, the synthesis ofthe block copolymers of the present invention begins with thepreparation of the PAA/BMA/DMAEMA hydrophobic block, and thehydrophilic, charged block is added in the second synthetic step eitherby subjecting the resulting PAA/BMA/DMAEMA macroCTA to a second RAFTpolymerization step. Alternate approaches include reducing thePAA/BMA/DMAEMA macroCTA to form a thiol end and then covalentlyattaching a pre-formed hydrophilic, charged polymer to the formed thiol.This synthetic approach provides a method for introduction of a reactivegroup on the ω-end of the hydrophilic end of the polymeric chain thusproviding alternate approaches to chemical conjugation to the polymer.

The diblock copolymer P7, one example of a polymer of the presentinvention, consists of two blocks; one is poly(DMAEMA), which ishydrophilic and charged at physiological pH, and the other block is arandom copolymer of monomer units: hydrophobic (BMA) andionized/hydrophobic or ionizable/hydrophobic units (PAA, DMAEMA).

Definitions and Embodiments

It is understood that, with regard to this application, use of thesingular includes the plural and vice versa unless expressly stated tobe otherwise. That is, “a” and “the” refer to one or more of whateverthe word modifies. For example, “the polymer” or “a nucleotide” mayrefer to one polymer or nucleotide or to a plurality of polymers ornucleotides. By the same token, “polymers” and “nucleotides” would referto one polymer or one nucleotide as well as to a plurality of polymersor nucleotides unless, again, it is expressly stated or obvious from thecontext that such is not intended.

A used herein, words of approximation such as, without limitation,“about” “substantially,” “essentially” and “approximately” mean that theelement of limitation so modified need not be exactly that which iswritten but may vary from that written description to some extent. Theextent to which the description may vary will depend on how great achange one of ordinary skill in the art would accept and still considerthe element to have the characteristics and capabilities of that elementor limitation. In general, but subject to the preceding discussion, anumerical value herein that is modified by a word of approximation mayvary from the stated value by at least ±15%, about ±15%, about ±10%,about ±5%, about ±3%, about ±2%, or about ±1%. As a specificnon-limiting example from this invention, the second block of a diblockcopolymer of this invention, which contains both cationic and anionicspecies at normal physiological pH, is described as being “substantiallyneutral in overall charge” and substantially hydrophobic.Experimentally, however, it is extremely difficult to achieve exactneutrality and either the cationic or the anionic species maypredominate to some extent as illustrated in Table 1. One of ordinaryskill in the art would, however, accept a second block with a slightexcess of one or the other charged species as still being “substantiallyneutral.”

As used herein, a “polymer” refers to a molecule composed of one or moresmaller molecules called “monomers.” A monomer may react with itself tocreate a homopolymer or it may react with one or more other monomers tocreate copolymers. Groups of monomers may be reacted to form“prepolymers,” which are then combined to form the polymer. The monomerscomprise the “constitutional units” of the polymer.

A “charge neutral” or “non-charged” constitutional unit refers to one inwhich no atom bears a full positive or negative charge at physiologicalpH, that is, dipolar molecules are still considered “charge neutral” or“non-charged”. An non-limiting example of a charge neutralconstitutional unit would be that derived from butyl methacrylate,CH₂═C(CH₃)C(O)O(CH₂)₃CH₃ monomer.

As used herein, “alkyl” refers to a straight or branched chain fullysaturated (no double or triple bonds) hydrocarbon (carbon and hydrogenonly) group. Examples of alkyl groups include, but are not limited to,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiarybutyl, pentyl and hexyl. As used herein, “alkyl” includes “alkylene”groups, which refer to straight or branched fully saturated hydrocarbongroups having two rather than one open valences for bonding to othergroups. Examples of alkylene groups include, but are not limited tomethylene, —CH₂—, ethylene, —CH₂CH₂—, propylene, —CH₂CH₂CH₂—,n-butylene, —CH₂CH₂CH₂CH₂—, sec-butylene, —CH₂CH₂CH(CH₃)— and the like.An alkyl group of this invention may optionally be substituted with oneor more fluorine groups.

As used herein, “mC to nC,” wherein m and n are integers refers to thenumber of possible carbon atoms in the indicated group. That is, thegroup can contain from “m” to “n”, inclusive, carbon atoms. An alkylgroup of this invention may comprise from 1 to 10 carbon atoms, that is,m is 1 and n is 10. Of course, a particular alkyl group may be morelimited. For instance without limitation, an alkyl group of thisinvention may consist of 3 to 8 carbon atoms, in which case it would bedesignated as a (3C-8C)alkyl group. The numbers are inclusive andincorporate all straight or branched chain structures having theindicated number of carbon atoms. For example without limitation, a “C₁to C₄ alkyl” group refers to all alkyl groups having from 1 to 4carbons, that is, CH₃—, CH₃CH₂—, CH₃CH₂CH₂—, CH₃CH(CH₃)—, CH₃CH₂CH₂CH₂—,CH₃CH₂CH(CH₃)—, (CH₃)₂CHCH₂— and (CH₃)₃CH—.

As use herein, a cycloalkyl group refers to an alkyl group in which theend carbon atoms of the alkyl chain are covalently bonded to oneanother. The numbers “m” and “n” refer to the number of carbon atoms inthe ring formed. Thus for instance, a (3C-8C) cycloalkyl group refers toa three, four, five, six, seven or eight member ring, that is,cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane andcyclooctane. A cycloalkyl group of this invention may optionally besubstituted with one or more fluorine groups and/or one or more alkylgroups.

As used herein, “phenyl” simply refers to a

group which, as shown, can optionally be substituted with one or morefluorine groups.

As used herein, a “hydrophobicity-enhancing moiety” is usedinterchangeably herein with a “hydrophobic species” and refers to asubstituent covalently bonded to a constitutional unit of a diblockcopolymer, with such constitutional units bearing saidhydrophobicity-enhancing moieties resulting in the diblock copolymerbecoming more membrane disruptive or otherwise more membranedestabilizing than it would be without the addition of the moiety.Examples of such moieties include, without limitation, alkyl groups,cycloalkyl groups and phenyl groups, any of which may be substitutedwith one or more fluorine atoms. In some embodiments, ahydrophobicity-enhancing moiety has a π value of about one, or more. Acompound's π value is a measure of its relative hydrophilic-lipophilicvalue (see, e.g., Cates, L. A., “Calculation of Drug Solubilities byPharmacy Students” Am. J. Pharm. Educ. 45:11-13 (1981)). Hydrophobicmonomeric residues or constitutional units described herein comprise oneor more hydrophobic species. Moreover, hydrophilic monomeric residuescomprise one or more hydrophilic species.

With regard to the non-limiting exemplary polymer of this invention IV1shown above, such a polymer would be characterized as being “ethylenic”in that the constitutional units are derived from the reaction of anethylene, —C═C—, functionality of each of the monomers. The particularethylenic monomers of the above example may be further described asbeing “acrylic” in that they are all derivatives of acrylic acid,CH₂═CHC(O)OH, the first monomer above being dimethylaminoethylmethacrylate, the second being 2-propylacrylic acid and the third beingbutyl methacrylate.

As used herein, “normal physiological pH” refers to the pH of thepredominant fluids of the mammalian body such as blood, serum, thecytosol of normal cells, etc. Moreover, as used herein, “normalphysiological pH”, used interchangeably with “about physiologic pH” or“about neutral pH”, generally refers to an about neutral pH (i.e., aboutpH 7), including, e.g., a pH that is about 7.2 to about 7.4. In specificinstances, a “normal physiological pH” refers to a pH that is aboutneutral in an aqueous medium, such as blood, serum, or the like.

As used herein, RNA refers to a polynucleotide comprising A, C, G or Unucleotides and DNA refers to a polynucleotide comprising dA, dC, dG anddT, the “d” indicating that the sugar is deoxyribose.

As used herein, a “natural DNA analog” or a “natural RNA analog” apolynucleotide in which one or more naturally-occurring nucleotides aresubstituted for the natural nucleotides of a particular DNA or RNA butwhich still exhibits the functionality of the original DNA or RNA. Thisincludes a naturally-occurring nucleotide in a non-natural environment,e.g., a ribonucleotide substituted for a deoxyribonucleotide in a DNAmolecule or a deoxyribonucleotides substituted for a ribonucleotide inan RNA molecule.

As used herein, a “synthetic DNA analog” or a “synthetic RNA analog”refers to a polynucleotide comprised of one or more modifiednucleotides. A “modified nucleotide” refers to a non-naturally occurringnucleotide that comprises a chemically altered base, sugar and/orphosphodiester linkage. Chemical alteration may involve addition,deletion or substitution of individual atoms of a naturally-occurringnucleotide or the addition, deletion of substitution of entirefunctional groups of the nucleotide. For the purposes of this inventiona modified nucleotide may indeed comprise a molecule that resembles anatural nucleotide little, if at all, but is nevertheless capable ofbeing incorporated into a polynucleotide having the generic structuredescribed above. One property of a synthetic DNA or RNA analog that istypically maintained is that the molecule is generally negativelycharged as are all natural polynucleotides so that it can complex with adiblock copolymer of this invention.

Without being bound by theory not expressly recited in the claims, amembrane destabilizing polymer can directly or indirectly elicit achange (e.g., a permeability change) in a cellular membrane structure(e.g., an endosomal membrane) so as to permit an agent (e.g.,polynucleotide), in association with or independent of a polymer, topass through such membrane structure—for example to enter a cell or toexit a cellular vesicle (e.g., an endosome). A membrane destabilizingpolymer can be (but is not necessarily) a membrane disruptive polymer. Amembrane disruptive polymer can directly or indirectly elicit lysis of acellular vesicle or disruption of a cellular membrane (e.g., as observedfor a substantial fraction of a population of cellular membranes).

Generally, membrane destabilizing or membrane disruptive properties ofpolymers can be assessed by various means. In one non-limiting approach,a change in a cellular membrane structure can be observed by assessmentin assays that measure (directly or indirectly) release of an agent(e.g., polynucleotide) from cellular membranes (e.g., endosomalmembranes)—for example, by determining the presence or absence of suchagent, or an activity of such agent, in an environment external to suchmembrane. Another non-limiting approach involves measuring red bloodcell lysis (hemolysis)—e.g., as a surrogate assay for a cellularmembrane of interest. Such assays may be done at a single pH value orover a range of pH values.

It is preferred that a diblock copolymer provided herein isbiocompatible. As used herein, “biocompatible” refers to a property of apolymer characterized by it, or its in vivo degradation products, beingnot, or at least minimally and/or reparably, injurious to living tissue;and/or not, or at least minimally and controllably, causing animmunological reaction in living tissue. With regard to salts, it ispresently preferred that both the cationic and the anionic species bebiocompatible. As used herein, “physiologically acceptable” isinterchangeable with biocompatible.

In certain aspects, the compositions and/or agents described herein areused as in vivo therapeutic agents. By “in vivo” is meant that they areintended to be administered to subjects in need of such therapy.“Subjects” refers to any living entity that might benefit from treatmentusing the complexes of this invention. As used herein “subject” and“patient” may be used interchangeably. A subject or patient refers inparticular to a mammal such as, without limitation, cat, dog, horse,cow, sheep, rabbit, etc., and preferably at present, a human being.

As used herein, “therapeutic agent” refers to a complex hereof that,when administered in a therapeutically effective amount to a subjectsuffering from a disease, has a therapeutic beneficial effect on thehealth and well-being of the subject. A therapeutic beneficial effect onthe health and well-being of a subject includes, but is not limited to:(1) curing the disease; (2) slowing the progress of the disease; (3)causing the disease to retrogress; or, (4) alleviating one or moresymptoms of the disease. As used herein, a therapeutic agent alsoincludes any complex herein that when administered to a patient, knownor suspected of being particularly susceptible to a disease inparticular at present a genetic disease, has a prophylactic beneficialeffect on the health and well-being of the patient. A prophylacticbeneficial effect on the health and well-being of a patient includes,but is not limited to: (1) preventing or delaying on set of the diseasein the first place; (2) maintaining a disease at a retrogressed levelonce such level has been achieved by a therapeutically effective amountof the complex; or, (3) preventing or delaying recurrence of the diseaseafter a course of treatment with a therapeutically effective amount ofthe complex has concluded. In some instances, a therapeutic agent is atherapeutically effective polynucleotide (e.g., an RNAi polynucleotide),a therapeutically effective peptide, a therapeutically effectivepolypeptide, or some other therapeutically effective biomolecule. Inspecific embodiments, an RNAi polynucleotide is an polynucleotide whichcan mediate inhibition of gene expression through an RNAi mechanism andincludes but is not limited to messenger RNA (mRNA), siRNA, microRNA(miRNA), short hairpin RNA (shRNA), asymmetrical interfering RNA(aiRNA), dicer substrate and the precursors thereof.

As used herein, “living polymerization” refers to a method ofsynthesizing polymers using the well-known concept of additionpolymerization, that is, polymerization wherein monomers are addedone-by-one to an active site on the growing polymer chain but onewherein the active sites for continuing addition of another monomer arenever fully eliminated other than on purpose. That is, the polymer chainis virtually always capable of further extension by the addition of moremonomer to the reaction mixture unless the polymer has been capped,which may be reversible so as permit polymerization to continue orquenched, which is usually permanent. While numerous genera of livingpolymerizations are known, currently the predominant types are anionic,cationic and radical living polymerizations. Of these, at presentradical polymerization is of particular interest with regard to thisinvention. Radical polymerization involves a free radical initiator thatextracts one of the pi electrons of the double bond of an ethylenicmonomer resulting in a reactive unpaired electron on the carbon at theother end of the former double bond from that with which the initiatorreacted. The unpaired electron then reacts with the double bond ofanother monomer creating a stable sigma bond and another free radicaland so on. With conventional initiators the sequence is eventuallystopped by a termination reaction, generally a combination reaction inwhich the unpaired electrons of two propagating chains combine to form astable sigma bond or a disproportionation in which a radical on a activechain strips a hydrogen atom from another active chain or from animpurity in the reaction mixture to produce a stable unreactive moleculeand a molecule containing a double bond. In a living polymerization theability of the growing chains to enter into a termination reaction iseliminated, effectively limiting the polymerization solely by the amountof monomer present; that is, the polymerization continues until thesupply of monomer has been exhausted. At this point the remaining freeradical species become substantially less active due to capping of thefree radical end group with such entities as, without limitation,nitroxyl radicals, halogen molecules, oxygen species such as peroxideand metals or simply by interaction with solvent and the like. If,however, more monomer is added to the solution, the polymerizationreaction can resume except as noted above.

Synthesis

Polymers described herein can be prepared in any suitable manner. Forexample, in certain embodiments, wherein polymers of this invention,while being in no way limited to ethylenic species, with regard to suchpolymers, it presently particularly preferred that they be prepared by“living polymerization.”

Using living polymerization, polymers of very low polydispersity ordifferences in chain length can be obtained. Polydispersity is usuallymeasured by dividing the weight average molecular weight of the polymerchains by their number average molecular weight. The number averagemolecule weight is sum of individual chain molecular weights divided bythe number of chains. The weight average molecular weight isproportional to the square of the molecular weight divided by the numberof molecules of that molecular weight. Since the weight averagemolecular weight is always greater than the number average molecularweight, polydispersity is always greater than or equal to one. As thenumbers come closer and closer to being the same, i.e., as thepolydispersity approaches a value of one, the polymer becomes closer tobeing monodisperse in which every chain has exactly the same number ofconstitutional units. Polydispersity values approaching one areachievable using radical living polymerization. Methods of determiningpolydispersity such as, without limitation, size exclusionchromatography, dynamic light scattering, matrix-assisted laserdesorption/ionization chromatography and electrospray masschromatography are well known in the art and will not be furtherdescribed herein.

Reversible addition-fragmentation chain transfer or RAFT is a presentlypreferred living polymerization technique for use in synthesizingethylenic backbone polymer of this invention. RAFT is well-known tothose skilled in the art and will only briefly be described herein. RAFTcomprises a free radical degenerative chain transfer process. Most RAFTprocedures employ thiocarbonylthio compounds such as, withoutlimitation, dithioesters, dithiocarbamates, trithiocarbonates andxanthates to mediate polymerization by a reversible chain transfermechanism. Reaction of a polymeric radical with the C═S group of any ofthe preceding compounds leads to the formation of stabilized radicalintermediates. These stabilized radical intermediates do not undergo thetermination reactions typical of standard radical polymerization but,rather, reintroduce a radical capable of re-initiation or propagationwith monomer, reforming the C═S bond in the process. This cycle ofaddition to the C═S bond followed by fragmentation of the ensuingradical continues until all monomer has been consumed or the reaction isquenched. The low concentration of active radicals at any particulartime limits normal termination reactions. In other embodiments, polymersare synthesized by Macromolecular design via reversibleaddition-fragmentation chain transfer of Xanthates (MADIX) (DirectSynthesis of Double Hydrophilic Statistical Di- and Triblock CopolymersComprised of Acrylamide and Acrylic Acid Units via the MADIX Process”,Daniel Taton, et al., Macromolecular Rapid Communications, 22, No. 18,1497-1503 (2001).)

Polymer:Biomolecule Constructs

Provided in certain embodiments herein are polymer:polynucleotideconstructs, or other constructs including, e.g., polymer:peptideconstructs, polymer:polypeptide constructs, or other types ofpolymer:biomolecule constructs. In certain embodiments, one or morepolynucleotide (e.g., siRNA) is associated with any polymer describedherein. In various embodiments, polynucleotide, peptides, polypeptides,or other biomolecules are conjugated to the polymer in any suitablemanner (e.g., by covalent and/or non-covalent interactions), and suchconjugation is at any suitable location, including, e.g., at the alphaend of the polymer, the omega end of the polymer, the hydrophilic end ofthe polymer, the hydrophobic end of the polymer, or to a pendant groupattached to a monomer side chain of the polymer.

As used herein, a polynucleotide refers to a member of the genus oforganic polymer molecules comprised of a linear chain of nucleotidemonomers covalently bonded in a chain, as such are well-known to thoseskilled in the art. In brief, a nucleotide comprises a nucleoside thatlinked to a single phosphate group (or, by convention, when referring toits incorporation into a polynucleotide, a short-hand for a nucleosidetriphosphate which is the species that actually undergoes polymerizationin the presence of a polymerase). A nucleoside, in turn, comprises abase linked to a sugar moiety. For naturally-occurring polynucleotides,i.e., polynucleotides produced by unmodified living entities, the sugarmoiety is either ribose, which gives rise to ribonucleic acids or RNAsor deoxyribose, which gives rise to deoxyribonucleic acids or DNA. Thenaturally-occurring bases are adenine (A), guanine (G) or its naturalsubstitute inosine (I), cytosine (C) or thymine (T) or its naturalsubstitute uracil (U). A polynucleotide, then, comprises a plurality ofnucleosides connected by a phosphodiester linkage between the3′-hydroxyl group of the sugar moiety of one nucleoside and the5′-hydroxyl of the sugar moiety of a second nucleoside which in turn islinked through its 3′-hydroxyl to the 5′- of yet another nucleoside andso on.

A DNA or an RNA of this invention may be sense or antisense. DNA isdouble-stranded, one strand being the sense strand and the other beingits complement or antisense strand. The sense strand is characterized bythe fact that an RNA version of the same sequence can be translated intoa protein. The antisense strand cannot participate in the same sequence.The consequence of this is that protein production by a particular DNAor its messenger RNA can be interrupted by introducing a complementaryor antisense polynucleotide at the appropriate stage of proteinproduction.

In brief, protein production occurs in two phases, transcription andtranslation. In transcription, DNA is used as a template to createmessenger RNA or mRNA. In the translation phase, the mRNA travels to aregion of the cell where it communicates the genetic message provided bythe DNA to the ribosome, which is the cellular machinery that actuallyassembles the protein encoded for by the DNA. An antisensepolynucleotide, which comprises a nucleic acid sequence that iscomplementary to that of an mRNA can bind or hybridize to the mRNA andthe hybridized mRNA is subsequently degraded by one or more biochemicalmechanisms thereby preventing the mRNA's instructions from reaching theribosome. It is presently preferred that a polynucleotide of thisinvention be an RNA.

The RNA of this invention may be sense or antisense mRNA, micro or miRNAor short interfering RNA, siRNA. mRNA is discussed above. miRNAs aresingle stranded RNA molecules about 21-23 nucleotides in length. Theirfunction is to regulate gene expression. miRNAs are encoded by genesthat are transcribed from DNA but are not translated into protein.Rather, they are processed from primary transcripts known a pri-miRNA toshort stem-loop structures called pre miRNA and finally to functionalmiRNA. Functional miRNAs are partially complementary to one or moremRNAs. As such, they perform like the antisense polynucleotidesdiscussed above and prevent mRNAs instructions from reaching theribosome. They thus are capable of down-regulating gene expression.

While miRNAs are transcribed from the genome itself, siRNAs, smallinterfering or short interfering RNAs, are not. siRNA, since itsdiscovery in 1999, has become one of the most studied polynucleotides inthe molecular biologists' arsenal and is currently considered a primecandidate for a next generation of drugs, since they are potentiallyable to silence the expression of virtually any gene. For the purposesof this invention, any siRNA currently known or as may become known inthe future can be used to form complexes of this invention with thediblock copolymers herein and thereupon can be transported into theinterior of living cells for, without limitation, therapeutic,prophylactic or diagnostic purposes. Initially it was thought thatexogenously added siRNAs had to be of a specific length (21 23 bp) withvery specific 2-base overhangs to be active as siRNAs, but it is nowclear that longer or shorter blunt-ended, as well as 27+bp RNAs are justas effective at gene silencing in mammalian cells. The shorter siRNAscan be loaded directly into the RNA induced silencing complex (RISC),while longer double-stranded RNAs can be cleaved by the cytoplasmicmultidomain endonuclease Dicer into shorter siRNAs in the cytoplasm. Inbrief, long double-stranded RNA enters the cytoplasm of a cell. The longdouble stranded RNA is processed into 20 to 25 nucleotide siRNAs by anRNase III-like enzyme called Dicer. The siRNA then assemble intoendoribonuclease-containing complexes known as RNA-induced silencingcomplex or RISC. After integration into the RISC, the sense strand ofthe double-stranded siRNAs is unwound and/or cleaved leaving the siRNAantisense strand which guides the RISC to a complementary mRNA molecule.The siRNA then binds to the complementary mRNA and once bound, the RISCcleaves the target mRNA, effectively silencing the gene associated withthat RNA. Another subgenus of polynucleotides that may form complexeswith diblock copolymers of this invention and thereby transported intoliving cells are the so-called “locked nucleic acid” or LNApolynucleotides. Locked nucleic acid polynucleotides may be prepared bya number of mechanisms one of which is the formation of a 2′-oxygen to4′-carbon methylene linkage in the sugar moiety of a nucleoside;however, use of any locked nucleic acid polynucleotide is within thescope of this invention. One characteristic of LNAs is their enhancedthermal stability when hybridized with complementary DNAs or RNAscompared to unmodified DNA:DNA or DNA:RNA duplexes as well as enhancednucleic acid recognition. These properties make LNA polynucleotidespotentially useful in a host of molecular application. For example, acomparison of an LNA-DNA-LNA construct with siRNA, phosphorothioate and2′-O-methyl RNA DNA constructs against expression of vanilloid receptorsubtype 1 (VR1) in Cos-7 cells revealed that, while siRNA were the mostpotent antisense agents against VR1 expression, the LNA-DNA-LNAconstruct was 175- and 550-fold more potent in suppressing VR1 thanisosequential phosphorothioate and 2′O-methyl oligonucleotides.Grunweller, A., et al., 2003, NAR, 31:2185-3193.

An aspect of this invention is a polynucleotide or a plurality ofpolynucleotides that are attached to or associated with (e.g., in acovalent and/or non-covalent manner, including ionic interactions,hydrogen-bonding interactions, and/or van der Waals interactions) anypolymer described herein. In certain embodiments, the associationbetween the polymer and polynucleotide is achieved by covalent bonds,non-covalent interactions, or combinations thereof. In specificembodiments, non-covalent associations of the polymer (e.g., of thefirst block thereof) with the polynucleotide are used. Non-covalentinteractions include, without limitation, ionic interactions, hydrogenbonding and van der Waals forces but for the purposes of the currentinvention the non-covalent interaction comprises ionic interactions. Theionic interaction arises between the cationic constitutional unit of apolymer (e.g., of the first block thereof) and the polynucleotide, whichis naturally negatively charged by virtue of the phosphodiesterlinkages:

Non-covalent association may be achieved by several additional methods.The polynucleotides and/or the polymer may be modified with chemicalmoieties that lead them to have an affinity for one another, such as alinkage, arylboronic acid-salicylhydroxamic acid, leucine zipper orother peptide motifs, ionic interactions between positive and negativecharges on the polymer and polynucleotide, or other types ofnon-covalent chemical affinity linkages. Additionally, a double-strandedpolynucleotide can be complexed to a polymer of the present invention byforming a polymer with a minor groove binding or an intercalating agentcovalently attached to the polymer.

In some embodiments, the polynucleotide may be chemically conjugated tothe polymer by any standard chemical conjugation technique. The covalentbond between the polymer and the polynucleotide may be non-cleavable, orcleavable bonds may be used. Particularly preferred cleavable bonds aredisulfide bonds that dissociate in the reducing environment of thecytoplasm. Covalent association is achieved through chemical conjugationmethods, including but not limited to amine-carboxyl linkers, aminesulfhydryl linkers, amine-carbohydrate linkers, amine-hydroxyl linkers,amine-amine linkers, carboxyl-sulfhydryl linkers, carboxyl-carbohydratelinkers, carboxyl hydroxyl linkers, carboxyl-carboxyl linkers,sulfhydryl-carbohydrate linkers, sulfhydryl-hydroxyl linkers,sulfhydryl-sulfhydryl linkers, carbohydrate-hydroxyl linkers,carbohydrate-carbohydrate linkers, and hydroxyl-hydroxyl linkers.Conjugation can also be performed with pH sensitive bonds and linkers,including, but not limited to, hydrazone and acetal linkages. A largevariety of conjugation chemistries are established in the art (see, forexample, Bioconjugation, Aslam and Dent, Eds, Macmillan, 1998 andchapters therein). Polynucleotides may be conjugated to the alpha oromega ends of the polymer, or to the pendant groups on the polymermonomers.

A series of polymers and their respective siRNA-condensed particles arecharacterized for size and surface charge and the resulting data areshown in Table 2.

In certain instances, polymers appear unimeric (<10 nm) in solution.Complexes formed from polymers and siRNA at theoretical charge ratios of4:1 ranged in sizes from 85-236 nm. There seemed to be no definitivetrend for the complex sizes with respect to BMA content. However,polymer P7 with 48% BMA content in the endosomolytic block exhibited thesmallest particle size of 85 nm±0.20. The remainder of the particles hadsizes from 144 to 236 nm, where the greatest sized particles were formedfrom polymer 6 which had 27% BMA content in the endosomolytic block.Polymer P7/siRNA particle sizes were further examined with charge ratiosranging from 1:1 to 8:1, and data are shown in Table 3. Polymer/siRNAparticle sizes decrease dramatically as charge ratio increases withvalues of 643 nm±0.09 at 1:1 to 54 nm±0.27 at 8:1.

TABLE 2 Size and ζ-potential measurements of particles formulated withsiRNA at a theoretical charge ratio of 4:1 as a function of butylmethacrylate composition. Diameter Zeta Potential Standard Polymer #(nm) PDI (mV) Error P1 166 0.14 1.1 1.32 P2 189 0.09 0.13 0.69 P3 1970.06 0.47 0.59 P4 144 0.11 0.41 1.2 P5 193 0.32 0.52 0.77 P6 236 0.060.67 0.95 P7 85 0.20 0.18 1.0

TABLE 3 Size and ζ-potential measurements of particles formulated withpolymer 7, the composition with the greatest butyl content, and siRNA asa function of charge ration. Theoretical Charge Ratio Diameter ZetaPotential Standard (+/−) (nm) PDI (mV) Error 1:1 643 0.09 0.27 1.1 2:1530 0.16 0.99 0.91 4:1 85 0.2 0.18 1.01 8:1 54 0.27 0.41 0.81

In certain instances, surface charge of siRNA/polymer complexes, basedon ζ-potential measurements, is found to be similar and slightlypositive for all polymers (˜0.5 mV with a range of 0.13-1.1 mV).Moreover, in some instances, complexes formed at +/− of 1:1, 2:1, 4:1,and 8:1 using polymer 7 showed no difference in surface charges, againwith slightly positive values (0.18-0.99 mV) with no trend with respectto charge ratio. In some instances, at 1:1 charge ratios, particles areexpected to have very little surface charge, as the PAA and DMAEMAcharges in the second block counterbalance each other. In variousinstances, as the charge ratio increases to 2:1, 4:1, and 8:1, one wouldexpect to see increases in positive surface charge, but interestingly,such was not observed. In some instances, with increasing amounts ofpolymer, the particles change morphology, becoming more tightly packed.With increasing charge ratio, it is possible that the surface charge isunaffected due to effective shielding of the DMAEMA positive charges, asmany polymer chains and siRNA become packed within the core of theparticles.

In certain embodiments, the alterations in particle size and surfacecharge may be relevant design criteria with regard to complex uptake bya cell. In some instances, nanoparticles bearing a positive surfacecharge facilitate uptake by electrostatic interactions with negativelycharged cell membranes.

Both polymer and siRNA/polymer complexes were evaluated for theirability to induce red blood cell hemolysis at pH values relevant to theendosomal/lysosomal trafficking pathway. Significant hemolysis did notoccur for polymers 1-3. However, relevant pH-dependent hemolyticactivity was evident with polymer 4, and enhanced responsiveness wasfound as BMA content of the endosomolytic block increased. Polymer 7exhibited the greatest pH-dependent hemolysis with essentially noactivity at pH=7.4, about 25% hemolysis at pH=6.6, and 85% hemolysis atpH=5.8. Polymers 5-7 were subsequently evaluated for hemolytic activityin their siRNA-complexed form. Complexes formed with polymers 5-7 at allcharge ratios tested were found to be hemolytic in a relevantpH-dependent fashion. Moreover, the hemolysis exhibited by complexes wasincreased when compared with free polymer and was greater at a chargeratio of 4:1 versus 1:1. Polymer 7 showed the greatest hemolyticactivity at a charge ratio of 4:1, with essentially no hemolysis atpH=7.4, 60% hemolysis at pH=6.8, and 100% hemolysis at pH 5.8. Thesedata suggest that the pH-responsive hemolytic activity of these polymersis linked to the incorporation of the hydrophobicity enhancing moiety,butyl methacrylate. This finding corroborates previous reports onpH-responsive, membrane destabilizing polymers that have utilizedincorporation of hydrophobic moieties such as alkyl amines or aromaticgroups to enhance the pH-dependent hydrophobic transition of carboxylatefunctionalized polymers.

Membrane Disruption and/or Membrane Destabilization

In certain embodiments, a polymer or polymer: polynucleotide construct(i.e., comprising any polymer described herein associated with one ormore polynucleotide) is a cellular membrane destabilizing or disruptivepolymer (i.e., is destabilizing or disruptive of a cellular membrane).In certain embodiments, the cellular membrane is, by way of non-limitingexample, an extracellular membrane, an intracellular membrane, avesicle, an organelle, an endosome, a liposome, or a red blood cell. Insome embodiments, when administered to a cell, the membrane disruptivepolymer or polymer:polynucleotide is delivered into the cell. In certainembodiments, siRNA is a preferred polynuceotide to be associated with apolymer of this invention and subsequently endocytosed with the polymerinto the interior of living cells

Endocytosis is the process by which a substance (for example, a polymer,or nucleic acid of the present invention) gains entrance into a cellwithout having to traverse the plasma membrane. The substance isenveloped by a portion of the cell membrane which then is pinched offforming an intracellular vesicle. Once the substance has beenendocytosed and the endosome has acidified, the chemical composition ofthe polymer is altered because the pKa of the polymer is selected suchthat, at the pH within a mature endosome, approximately 5-6.5, theequilibrium between the un-ionized and the ionized forms of the acidicunits, i.e., the anionic constitutional units of a polymer of thisinvention, is shifted to the un-ionized form. In contrast to the ionizedform of the polymer, which is relatively hydrophilic, the un ionizedform is substantially hydrophobic and capable of interaction, i.e.,disruption of, the endosomal membrane which results in the release ofthe substance into the cytosol.

Cellular internalization of siRNA complexes at 4:1 charge ratios wasinvestigated using flow cytometry for polymers P4-P7 based on theirrelevant pH-responsive endosomolytic characteristics. Following 4 hoursof exposure to 25 nM of polymer-complexed siRNA, cellular uptake wasfound to positively correlate with BMA content of the second block, withpolymer P7 showing the highest level of uptake (23% siRNA positivecells) during this timeframe. Positively charged complexes have beenpreviously demonstrated to affect internalization of cationicpolymer/nucleic acid complexes, with positively charged complexesachieving higher internalization rates and transgene expression. Theseresults are likely not a function of surface charge or size, as all theparticles exhibit the same, slightly positive net charge and sizes(85-236 nm) well within the limits for non-specific endocytosis (Table2). Rather, the effect on uptake may be a function of the endosomolyticeffectiveness of the BMA-containing block. Based on hemolysis results,as BMA content increases, endosomal escape occurs to a greater extent,thus recycling from the cell decreases and net accumulation of siRNAwithin the cell increases, similar to other propylacrylicacid-containing bioconjugates. Based on electrostatic repulsion betweensiRNA and cell membranes, all polymer formulations showed much greateruptake (up to 25×) by cells than siRNA not complexed with a carrier(naked siRNA). Internalization of complexed siRNA by up to 23% of cellsonly after 4 hours is extremely promising for therapeutic efficacy, asthe cumulative uptake is likely to be much higher after the full 48 hourtreatment. In addition, siRNA activity is considered to be catalytic; itcan be recycled within the cytoplasm to destroy multiple mRNAtranscripts, therefore having a long-term, multi-generational effect.

The nonspecific cytotoxicity of the polymer carriers was investigated byincubating HeLa cells in the presence of the complexes at charge ratiosof 4:1 for 24 hours. High relative survival was observed (>90% after 24hours) for all polymers tested. Synthetic polymers, in particularcationic polymers, can be associated with appreciable cytotoxicity. Forinstance, PEI has been shown to trigger apoptosis and/or necrosis in avariety of cell lines. This toxicity can be reduced by chemicallymodifying the polycation segment with hydrophilic segments; however,there is usually a tradeoff between efficacy and toxicity. In thisapproach, the use of a charge-neutralized second block of the polymerdelivery vehicle presumably maintained high survivability of in vitrocultured HeLa cells.

The ability of the carriers to effectively deliver siRNA wasinvestigated with knockdown experiments against GAPDH with complexesformed from all polymers at theoretical charge ratios of 4:1. GAPDHprotein levels were evaluated 48 hours after treatment with thecomplexes. Polymer carriers 1-3 were ineffectual at eliciting reductionof protein levels, likely due to their inability to mediate endosomalescape. However, GAPDH protein reduction became evident with the use ofpolymer 4 as a siRNA carrier. The knockdown of protein further increasedas the BMA content of the carriers increased to 48% of the endosomolyticblock (polymer P7). Polymer P7 showed the greatest ability to mediatesiRNA knockdown of protein where GAPDH was reduced to 32% of control.Furthermore, control siRNA showed negligible reduction in GAPDH proteinlevels.

To further characterize carrier efficacy, polymers were analyzed fortheir ability to knockdown GAPDH mRNA levels. Similar to the proteinmeasurements, polymers 1-3 elicited very little reduction of mRNAsignal, as evaluated by RT-PCR. Again, polymers P4-P7 showed increasedknockdown of GAPDH as the BMA content of the endosomolytic blockincreased. Specifically, GAPDH knockdown was reduced to 39%, 30%, 31%,and 21% of control at a charge ratio of 4:1, for polymers P4, P5, P6,and P7, respectively. Overall, the results are consistent with findingsfrom other groups exploring delivery strategies for DNA which have foundthat the addition of hydrophobic domains, specifically N-oleyl moieties,phenylalanine resides, and butyl methacrylate, as utilized here, enhancetransfection.

Further investigation into ability of P7, which includes the polymer ofTable 2 and similar structures (including various versions of P7, suchas P7v6, which is used interchangeably herein with PRx0729v6), tomediate gene knockdown was performed with respect to charge ratio andsiRNA dose. Alteration of theoretical charge ratios was found to greatlyaffect gene knockdown. GAPDH was reduced to 51%, 42%, 21%, and 14% ofcontrol levels with charge ratios of 1:1, 2:1, 4:1, and 8:1,respectively. Particularly at charge ratios of 4:1 and 8:1, geneknockdown was similar to the commercially available carrier HiPerFect,where GAPDH levels were reduced by over 80%. Importantly, the effects onGAPDH levels are specific to the siRNA that is delivered, as when acontrol siRNA is utilized at a charge ratio of 8:1, there is nosignificant effect on GAPDH levels. Altering the charge ratio may haveresulted in differing levels of condensation of the siRNA within thenanoparticles. DLS experiments indicated that increasing copolymercontent in the complexes resulted in more condensed particles (Table 3),and these functional studies suggest that more compact particles can beinternalized more efficiently or with increased siRNA bioavailability.These findings are consistent with previous reports indicating that morecompact DNA/polyethyleneimine and DNA/polylysine complexes internalizeat higher rates and achieve higher transfection efficiencies. Adose-response study using P7 at a charge ratio of 4:1 was conducted.Although there was little response in GAPDH gene expression at 1 nM or 5nM siRNA, expression was reduced to 77%, 21%, and 12% of control when 10nM, 25 nM, or 50 nM of siRNA was delivered using polymer 7. This levelof knockdown approaches that seen using 50 nM HiPerFect, a commerciallyavailable positive control. However, all polymers, including polymer 7,demonstrated enhanced biocompatibility, as measured by nonspecificcytotoxicity compared to HiPerFect. Although significant levels of geneknockdown with higher doses of siRNA (25-50 nM) were achieved, fortranslation to in vivo applications, it may be more desirable to achievesignificant reduction using lower doses of siRNA to avoid off-targeteffects. In some embodiments, this is accomplished by more efficientuptake of the polymer/siRNA particles, perhaps best accomplished bytargeting ligands.

Uses

In certain embodiments, the polynucleotides of this invention that areadministered to a subject and ultimately delivered into the subject'scells are preferably DNA, RNA or natural or synthetic analogs thereof.With regard to DNA/RNA and analogs thereof that are able to inhibitexpression of target genes, these include such species as, withoutlimitation, antisense polynucleotides, miRNA and siRNA.

Diseases that are optionally treated using polymers and/or polymer:polynucleotide complexes of this invention include, without limitation,pathogenic disorders, cancers, inflammatory diseases, enzymedeficiencies, inborn errors of metabolism, infections, auto-immunediseases, cardiovascular diseases, neurological, neurodegenerative,diseases, neuromuscular diseases, blood disorders and clottingdisorders.

The following examples are for illustration purposes and are not to beconstrued as limiting the invention. All publications recited herein arehereby incorporated by reference for the information to which thepublications are cited.

Examples

Throughout the description of the present invention, various knownacronyms and abbreviations are used to describe monomers or monomericresidues derived from polymerization of such monomers. Withoutlimitation, unless otherwise noted: “BMA” (or the letter “B” asequivalent shorthand notation) represents butyl methacrylate ormonomeric residue derived therefrom; “DMAEMA” (or the letter “D” asequivalent shorthand notation) represents N,N-dimethylaminoethylmethacrylate or monomeric residue derived therefrom; “Gal” refers togalactose or a galactose residue, optionally includinghydroxyl-protecting moieties (e.g., acetyl) or to a pegylated derivativethereof (as described below); HPMA represents 2-hydroxypropylmethacrylate or monomeric residue derived therefrom; “MAA” representsmethylacrylic acid or monomeric residue derived therefrom; “MAA(NHS)”represents N-hydroxyl-succinimide ester of methacrylic acid or monomericresidue derived therefrom; “PAA” (or the letter “P” as equivalentshorthand notation) represents 2-propylacrylic acid or monomeric residuederived therefrom, “PEGMA” refers to the pegylated methacrylic monomer,CH₃O(CH₂O)₇₋₈OC(O)C(CH₃)CH₂ or monomeric residue derived therefrom. Ineach case, any such designation indicates the monomer (including allsalts, or ionic analogs thereof), or a monomeric residue derived frompolymerization of the monomer (including all salts or ionic analogsthereof), and the specific indicated form is evident by context to aperson of skill in the art.

Example 1: Preparation of Di-Block Polymers and Copolymers

Di-block polymers and copolymers of the following general formula areprepared:

[A1_(x)-/-A2_(y)]_(n)-[B1_(x)-/-B2_(y)-/-B3_(z)]_(1-5n)

Where [A1-A2] is the first block copolymer, composed of residues ofmonomers A1 and A2

[B1-B2-B3] is the second block copolymer, composed of residues ofmonomers B1, B2, B3

x, y, z is the polymer composition in mole % monomer residue

n is molecular weight

Exemplary di-block copolymers: [DMAEMA]-[B-/-P-/-D]

[PEGMA_(w)]-[B-/-P-/-D]

[PEGMA_(W)-DMAEMA]-[B-/-P-/-D]

[PEGMA_(w)-MAA(NHS)]-[B-/-P-/-D]

[DMAEMA-/-MAA(NHS)]-[B-/-P-/-D]

[HPMA-/-PDSM]-[B-/-P-/-D]

Where:

-   -   B is butyl methacrylate    -   P is propyl acrylic acid    -   D is DMAEMA is dimethylaminoethyl methacrylate    -   PEGMA is polyethyleneglycol methacrylate where, for example,        w=4-5 or 7-8 ethylene oxide units)    -   MAA(NHS) is methylacrylic acid-N-hydroxy succinamide    -   HPMA is N-(2-hydroxypropyl) methacrylamide    -   PDSM is pyridyl disulfide methacrylate

These polymers represent structures where the composition of the firstblock of the polymer or copolymer is varied or chemically treated inorder to create polymers where the first block is neutral (e.g., PEGMA),cationic (DMAEMA), anionic (PEGMA-NHS, where the NHS is hydrolyzed tothe acid), ampholytic (DMAEMA-NHS, where the NHS is hydrolyzed to theacid), or zwiterrionic (for example,poly[2-methacryloyloxy-2′trimethylammoniumethyl phosphate]). Inaddition, the [PEGMA-PDSM]-[B-P-D] polymer contains a pyridyl disulfidefunctionality in the first block that can be reacted with a thiolatedsiRNA to form a polymer-siRNA conjugate.

Example 1.1: General Synthetic Procedures for Preparation of BlockCopolymers by RAFT

A. RAFT Chain Transfer Agent.

The synthesis of the chain transfer agent (CTA),4-Cyano-4-(ethylsulfanylthiocarbonyl) sulfanylpentanoic acid (ECT),utilized for the following RAFT polymerizations, was adapted from aprocedure by Moad et al., Polymer, 2005, 46(19): 8458-68. Briefly,ethane thiol (4.72 g, 76 mmol) was added over 10 minutes to a stirredsuspension of sodium hydride (60% in oil) (3.15 g, 79 mmol) in diethylether (150 ml) at 0° C. The solution was then allowed to stir for 10minutes prior to the addition of carbon disulfide (6.0 g, 79 mmol).Crude sodium S-ethyl trithiocarbonate (7.85 g, 0.049 mol) was collectedby filtration, suspended in diethyl ether (100 mL), and reacted withIodine (6.3 g, 0.025 mol). After 1 hour the solution was filtered,washed with aqueous sodium thiosulfate, and dried over sodium sulfate.The crude bis (ethylsulfanylthiocarbonyl) disulfide was then isolated byrotary evaporation. A solution of bis-(ethylsulfanylthiocarbonyl)disulfide (1.37 g, 0.005 mol) and 4,4′-azobis(4-cyanopentanoic acid)(2.10 g, 0.0075 mol) in ethyl acetate (50 mL) was heated at reflux for18 h. Following rotary evaporation of the solvent, the crude 4-Cyano-4(ethylsulfanylthiocarbonyl) sulfanylvpentanoic acid (ECT) was isolatedby column chromatography using silica gel as the stationary phase and50:50 ethyl acetate hexane as the eluent.

B. Poly(N,N-dimethylaminoethyl methacrylate) macro chain transfer agent(polyDMAEMA macroCTA).

The RAFT polymerization of DMAEMA was conducted in DMF at 30° C. under anitrogen atmosphere for 18 hours using ECT and2,2′-Azobis(4-methoxy-2.4-dimethyl valeronitrile) (V-70) (Wakochemicals) as the radical initiator. The initial monomer to CTA ratio([CTA]₀/[M]₀ was such that the theoretical M_(n) at 100% conversion was10,000 (g/mol). The initial CTA to initiator ratio ([CTA]d[I]_(n)) was10 to 1. The resultant polyDMAEMA macro chain transfer agent wasisolated by precipitation into 50:50 v:v diethyl ether/pentane. Theresultant polymer was redissolved in acetone and subsequentlyprecipitated into pentane (×3) and dried overnight in vacuo.

C. Block Copolymerization of DMAEMA, PAA, and BMA from a Poly(DMAMEA)macroCTA.

The desired stoichiometric quantities of DMAEMA, PAA, and BMA were addedto poly(DMAEMA) macroCTA dissolved in N,N-dimethylformamide (25 wt %monomer and macroCTA to solvent). For all polymerizations [M]d[CTA]_(o)and [CTA]_(o)/[I]_(o) were 250:1 and 10:1 respectively. Following theaddition of V70 the solutions were purged with nitrogen for 30 min andallowed to react at 30° C. for 18 h. The resultant diblock copolymerswere isolated by precipitation into 50:50 v:v diethyl ether/pentane. Theprecipitated polymers were then redissolved in acetone and subsequentlyprecipitated into pentane (×3) and dried overnight in vacuo. Gelpermeation chromatography (GPC) was used to determine molecular weightsand polydispersities (PDI, M_(w)/M_(n)) of both the poly(DMAEMA)macroCTA and diblock copolymer samples in DMF with respect to polymethylmethacrylate standards (SEC Tosoh TSK-GEL R-3000 and R-4000 columns(Tosoh Bioscience, Montgomeryville, Pa.) connected in series to aViscotek GPCmax VE2001 and refractometer VE3580 (Viscotek, Houston,Tex.). HPLC-grade DMF containing 1.0 wt % LiBr was used as the mobilephase. FIG. 1A summarizes the molecular weights and compositions of someof the RAFT synthesized polymers. (PRx0729v6 is used interchangeablywith P7v6 in this application and in various priority applications.)FIG. 1B summarizes the molecular weights, particle size and compositionsof some of the RAFT synthesized polymers.

Example 1.2. Preparation of Second Block (B1-B2-B3) Copolymerization ofDMAEMA, PAA, and BMA from a Poly(PEGMA) macroCTA

The desired stoichiometric quantities of DMAEMA, PAA, and BMA were addedto poly(PEGMA) macroCTA dissolved in N,N-dimethylformamide (25 wt %monomer and macroCTA to solvent). For all polymerizations [M]d[CTA]_(o)and [CTA]_(o)/[I]₀ were 250:1 and 10:1 respectively. Following theaddition of AIBN the solutions were purged with nitrogen for 30 min andallowed to react at 68° C. for 6-12 h (FIG. 2). The resulting diblockcopolymers were isolated by precipitation into 50:50 v:v diethylether/pentane. The precipitated polymers were then redissolved inacetone and subsequently precipitated into pentane (×3) and driedovernight in vacuo. Gel permeation chromatography (GPC) was used todetermine molecular weights and polydispersities (PDI, M_(w)/M_(n)) ofboth the poly(PEGMA) macroCTA and diblock copolymer samples in DMF usinga Viscotek GPCmax VE2001 and refractometer VE3580 (Viscotek, Houston,Tex.). HPLC-grade DMF containing 1.0 wt % LiBr was used as the mobilephase. NMR spectroscopy in CDCl₃ was used to confirm the polymerstructure and calculate the composition of the 2nd block. FIG. 2summarizes the synthesis of [PEGMA_(w)]-[B-P-D] polymer where w=7-8.FIGS. 3A, 3B and 3C summarize the characterization of[PEGMA_(w)]-[B-P-D] polymer where w=7-8.

Example 1.3. Preparation and Characterization of PEGMA-DMAEMACo-Polymers

Polymer synthesis was carried out using a procedure similar to thatdescribed in Examples 1.1 and 1.2. The ratio of the PEGM and DMAEMA inthe first block was varied by using different feed ratios of theindividual monomers to create the co-polymers described in FIG. 4.

Example 1.4. Preparation and Characterization of PEGMA-MAA(NHS)Co-Polymers

Polymer synthesis was performed as described in Examples 1.1 and 1.2(and summarized in FIG. 5), using monomer feed ratios to obtain thedesired composition of the 1st block copolymer. FIGS. 6A, 6B and 6Csummarize the synthesis and characterization of[PEGMA_(w)-MAA(NHS)]-[B-P-D] polymer where the co-polymer ratio ofmonomers in the 1st block is 70:30. NHS containing polymers can beincubated in aqueous buffer (phosphate or bicarbonate) at pH between 7.4and 8.5 for 1-4 hrs at room temperature or 37° C. to generate thehydrolyzed (acidic) form.

Example 1.5. Preparation and Characterization of DMAEMA-MAA(NHS)Co-Polymers

Polymer synthesis was performed as described in Examples 1.1 and 1.2,using monomer feed ratios to obtain the desired composition of the1^(st) block copolymer. FIGS. 7A, 7B and 7C summarize the synthesis andcharacterization of [DMAEMA-MAA(NHS)]-[B-P-D] polymer where theco-polymer ratio of monomers in the 1st block is 70:30. NHS containingpolymers can be incubated in aqueous buffer (phosphate or bicarbonate)at pH between 7.4 and 8.5 for 1-4 hrs at room temperature or 37° C. togenerate the hydrolyzed (acidic) form.

Example 2. Preparation and Characterization of HPMA-PDS(RNA) Co-PolymerConjugates for siRNA Drug Delivery

A. Synthesis of pyridyl disulfide methacrylate monomer (PDSMA).

The synthesis scheme for PDSMA is summarized in FIG. 8. Aldrithiol-2™ (5g, 22.59 mmol) was dissolved in 40 ml of methanol and 1.8 ml of AcOH.The solution was added as a solution of 2-aminoethanethiol.HCl (1.28 g,11.30 mmol) in 20 ml methanol over 30 min. The reaction was stirredunder N₂ for 48 h at R.T. After evaporation of solvents, the residualoil was washed twice with 40 ml of diethyl ether. The crude compound wasdissolved in 10 ml of methanol and the product was precipitated twicewith 50 ml of diethyl ether to get the desired compound 1 as slightyellow solid. Yield: 95%.

Pyridine dithioethylamine (1, 6.7 g, 30.07 mmol) and triethylamine (4.23ml, 30.37 mmol) were dissolved in DMF (25 ml) and pyridine (25 ml) andmethacryloyl chloride (3.33 ml, 33.08 mmol) was added slowly via syringeat 0 C. The reaction mixture was stirred for 2 h at R.T. After reaction,the reaction was quenched by sat. NaHCO₃ (350 ml) and extracted by ethylacetate (350 ml). The combined organic layer was further washed by 10%HCl (100 ml, 1 time) and pure water (100 ml, 2 times) and dried byMaSO₄. The pure product was purified by column chromatography (EA/Hex:1/10 to 2/1) as yellow syrup. Rf=0.28 (EA/Hex=1/1). Yield: 55%.

B. HPMA-PDSMA Co-Polymer Synthesis

The RAFT polymerization of N-(2-hydroxypropyl) methacrylamide (HPMA) andpyridyl disulfide methacrylate (typically at a 70:30 monomer ratio) isconducted in DMF (50 weight percent monomer:solvent) at 68° C. under anitrogen atmosphere for 8 hours using 2,2′-azo-bis-isobutyrylnitrile(AIBN) as the free radical initiator (FIG. 9). The molar ratio of CTA toAIBN is 10 to 1 and the monomer to CTA ratio is set so that a molecularweight of 25,000 g/mol would be achieved if at 100% conversion. Thepoly(HPMA-PDS) macro-CTA was isolated by repeated precipitation intodiethyl ether from methanol.

The macro-CTA is dried under vacuum for 24 hours and then used for blockcopolymerization of dimethylaminoethyl methacrylate (DMAEMA),propylacrylic acid (PAA), and butyl methacrylate (BMA). Equimolarquantities of DMAEMA, PAA, and BMA ([M]_(o)/[CTA]_(o)=250) are added tothe HPMA-PDS macroCTA dissolved in N,N-dimethylformamide (25 wt %monomer and macroCTA to solvent). The radical initiator AIBN is addedwith a CTA to initiator ratio of 10 to 1. The polymerization is allowedto proceed under a nitrogen atmosphere for 8 hours at 68° C. Afterwards,the resultant diblock polymer is isolated by precipitation 4 times into50:50 diethyl ether/pentane, redissolving in ethanol betweenprecipitations. The product is then washed 1 time with diethyl ether anddried overnight in vacuo.

C. siRNA Conjugation to HPMA-PDSMA Co-Polymer

Thiolated siRNA was obtained commercially (Agilent, Boulder, Colo.) as aduplex RNA with a disulfide modified 5′-sense strand. The free thiolform for conjugation is prepared by dissolving the lyophilized compoundin water and treated for 1 hour with the disulfide reducing agent TCEPimmobilized within an agarose gel. The reduced RNA (400 μM) was thenreacted for 24 hours with the pyridyl disulfide-functionalized polymerin phosphate buffer (pH 7) containing 5 mM ethylenediaminetetraaceticacid (EDTA) (FIG. 9).

The reaction of the pyridyl disulfide polymer with the RNA thiol creates2-pyridinethione, which can be spectrophotometrically measured tocharacterize conjugation efficiency. To further validate disulfideexchange, the conjugates are run on an SDS-PAGE 16.5% tricine gel. Inparallel, aliquots of the conjugation reactions are treated withimmobilized TCEP prior to SDS-PAGE to verify release of the RNA from thepolymer in a reducing environment. Conjugation reactions are conductedat polymer/RNA stoichiometries of 1, 2, and 5. UV spectrophotometricabsorbance measurements at 343 nm for 2-pyridinethione release are usedto measure conjugation efficiencies.

Example 3: siRNA/Polymer Complex Characterization

After verification of complete, serum-stable siRNA complexation viaagarose gel retardation, siRNA/polymer complexes were characterized forsize and zeta potential using a ZetaPALS detector (BrookhavenInstruments Corporation, Holtsville, N.Y., 15 mW laser, incidentbeam=676 nm). Briefly, polymer was formulated at 0.1 mg/ml in phosphatebuffered saline (PBS, Gibco) and complexes were formed by addition ofpolymer to GAPDH siRNA (Ambion) at the indicated theoretical chargeratios based on positively charged DMAEMA, which is 50% protonated atpH=7.4 and the negatively-charged siRNA. Correlation functions werecollected at a scattering angle of 90°, and particle sizes werecalculated using the viscosity and refractive index of water at 25° C.Particle sizes are expressed as effective diameters assuming alog-normal distribution. Average electrophoretic mobilities weremeasured at 25° C. using the ZetaPALS zeta potential analysis software,and zeta potentials were calculated using the Smoluchowsky model foraqueous suspensions.

Example 4: HeLa Cell Culture

HeLas, human cervical carcinoma cells (ATCC CCL-2), were maintained inminimum essential media (MEM) containing L-glutamine (Gibco), 1%penicillin-streptomycin (Gibco), and 10% fetal bovine serum (FBS,Invitrogen) at 37° C. and 5% CO2.

Example 5: pH-Dependent Membrane Disruption of Carriers andsiRNA/Polymer Complexes

Hemolysis was used to determine the potential endosomolytic activity ofboth free polymer and siRNA/polymer conjugates at pH values that mimicendosomal trafficking (extracellular pH=7.4, early endosome pH=6.6, andlate endosome pH=5.8). Briefly, whole human blood was collected invaccutainers containing EDTA. Blood was centrifuged, plasma aspirated,and washed three times in 150 mM NaCl to isolate the red blood cells(RBC). RBC were then resuspended in phosphate buffer (PB) at pH 7.4, pH6.6, or pH 5.8. Polymers (10 ug/ml) or polymer/siRNA complexes were thenincubated with the RBC at the three pH values for 1 hour at 37° C.Intact RBC were then centrifuged and the hemoglobin released intosupernatant was measured by absorbance at 541 nm as an indication ofpH-dependent RBC membrane lysis. FIG. 10A shows the hemolysis ofpolymers at a concentration of 10 μg/ml and FIG. 10B shows polymer/siRNAcomplexes of polymers 5-7 at theoretical charge ratios of 1:1 and 4:1.Hemolytic activity was normalized relative to a positive control, 1% v/vTriton X-100 and are representative data from a single experimentconducted in triplicate±standard deviation.

Example 6: Measurement of Carrier-Mediated siRNA Uptake

Intracellular uptake of siRNA/polymer complexes was measured using flowcytometry (Becton Dickinson LSR benchtop analyzer). HeLas were seeded at15,000 cells/cm2 and allowed to adhere overnight. FAM(5-carboxyfluorescine) labeled siRNA (Ambion) was complexed with polymerat a theoretical charge ratio of 4:1 for 30 min at room temperature andthen added to the plated HeLas at a final siRNA concentration of 25 nM.After incubation with the complexes for 4 h, the cells were trypsinizedand resuspended in PBS with 0.5% BSA and 0.01% trypan blue. Trypan bluewas utilized as previously described for quenching of extracellularfluorescence and discrimination of complexes that have been endocytosedby cells. 10,000 cells were analyzed per sample and fluorescence gatingwas determined using samples receiving no treatment and polymer notcomplexed with FAM labeled siRNA. FIG. 11 shows HeLa cellinternalization of FAM-labeled siRNA and polymer/siRNA complexes formedwith polymers 4-7 and delivered for 4 h. Data are from three independentexperiments conducted in triplicate with error bars representingstandard error of the mean (SEM).

Example 7: siRNA/Polymer Complex Cytotoxicity

siRNA/polymer complex cytotoxicity was determined using and lactatedehydrogenase (LDH) cytotoxicity detection kit (Roche). HeLa cells wereseeded in 96-well plates at a density of 12,000 cells per well andallowed to adhere overnight. Complexes were formed by addition ofpolymer (0.1 mg/ml stock solutions) to GAPDH siRNA at theoretical chargeratios of 4:1 and to attain a concentration of 25 nM siRNA/well.Complexes (charge ratio=4:1) were added to wells in triplicate. Aftercells had been incubated for 24 hours with the polymer complexes, themedia was removed and the cells were washed with PBS twice. The cellswere then lysed with lysis buffer (100 uL/well, 20 mM Tris-HCl, pH 7.5,150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodiumpyrophosphate, 1 mM (3-glycerophosphate, 1 mM sodium orthovanadate) for1 hour at 4° C. After mixing by pipetting, 20 uL of the cell lysate wasdiluted 1:5 in PBS and quantified for lactate dehydrogenase (LDH) bymixing with 100 μL of the LDH substrate solution. After a 10-20 minincubation for color formation, the absorbance was measured at 490 nmwith the reference set at 650 nm.

FIG. 12A shows nonspecific HeLa cytotoxicity and FIG. 12B shows GAPDHknockdown as a function of siRNA polymer carrier. HeLa cells weretransfected with siRNA against GAPDH at 25 nM using polymer/siRNAcomplexes formulated at theoretical charge ratios of 4:1. (A) After 24h, cell lysate was collected and assayed for lactate dehydrogenase, ameasure of cell viability, and data is shown relative to untreatedcells. (B) After 48 h, both protein (black) and mRNA levels (white) wereexamined using a GAPDH enzyme activity assay and RT-PCR, respectively,and data is shown relative to cells receiving no treatment. Data arefrom three independent experiments conducted in triplicate with errorbars representing standard deviation.

Example 8: Evaluation of GAPDH Protein and Gene Knockdown bysiRNA/Polymer Complexes

The efficacy of the series of polymers for siRNA delivery was screenedusing a GAPDH activity assay (Ambion). HeLas (12,000 cells/cm2) wereplated in 96-well plates. After 24 h, complexes (charge ratios=4:1) wereadded to the cells at a final siRNA concentration of 25 nM in thepresence of 10% serum. The extent of siRNA-mediated GAPDH proteinreduction was assessed 48 h post-transfection. As a positive control,parallel knockdown experiments were run using HiPerFect (Qiagen)following manufacturer's conditions. The remaining GAPDH activity wasmeasured as described by the manufacturer using the kinetic fluorescenceincrease method over 5 min and was calculated according to the followingequation: % remaining expression=fluorescence, GAPDH/fluorescence, notreatment, where fluorescence=fluorescence-5 min-fluoresecence 1 min.The transfection procedure did not significantly affect GAPDH expressionwhen a nontargeting sequence of siRNA was used. FIG. 13A shows GAPDHknockdown in HeLas measured via real time RT PCR 48 h after treatmentwith complexes as a function of charge ratio (1:1-8:1) and FIG. 13Bshows GAPDH knockdown in HeLas measured via real time RT-PCR 48 h aftertreatment with complexes as a function of siRNA dose (1-50 nM) withpolymer 7 as the carrier. Negative control siRNA #1 (Ambion) and acommercially available transfection reagent, HiPerFect (Qiagen), wereused as negative and positive controls, respectively.

After the initial screen to identify the carrier that produced the mostrobust siRNA-mediated GAPDH knockdown, real time reverse transcriptionpolymerase chain reaction (RT-PCR) was used to directly evaluate siRNAdelivery. After 48 hours of incubation with complexes as formed above,cells were rinsed with PBS. Total RNA was isolated using Qiagen'sQiashredder and RNeasy mini kit. Any residual genomic DNA in the sampleswas digested (RNase-Free DNase Set, Qiagen) and RNA was quantified usingthe RiboGreen assay (Molecular Probes) based on the manufacturer'sinstructions.

Reverse transcription was performed using the Omniscript RT kit(Qiagen). A 25 ng total RNA sample was used for cDNA synthesis and PCRwas conducted using the ABI Sequence Detection System 7000 usingpredesigned primer and probe sets (Assays on Demand, Applied Biosystems)for GAPDH and beta-actin as the housekeeping gene. Reactions (20 μltotal) consisted of 10 μL of 2× Taqman Universal PCR Mastermix, 1 μL ofprimer/probe, and 2 μL of cDNA, brought up to 20 μL with nuclease-freewater (Ambion). The following PCR parameters were utilized: 95° C. for90 s followed by 45 cycles of 95° C. for 30 s and 55° C. for 60 s.Threshold cycle (CT) analysis was used to quantify GAPDH, normalized tobeta-actin and relative to expression of untreated HeLas.

Example 9. Functional Design of Poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)]

FIG. 14 shows the polymer design for Poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)].Multifunctional properties were incorporated via RAFT polymer synthesisstrategies using a pyridyl disulfide end-functionalized CTA to form adiblock architecture designed to possess aqueous solubility andpH-dependent membrane destabilizing properties. The monomer chemicalfunctionalities highlighted in FIG. 14 were chosen in order to producethe desired properties for each polymer block. Importantly, module 3 wasdesigned to be near charge neutrality at physiologic pH (approximately50% DMAEMA protonation and 50% PAA deprotonation predicted) and toundergo a transition to a more hydrophobic and positively charged statein lower pH environments.

Example 10. Synthesis of Pyridyl Disulfide-CTA

The 4-cyano-4-(ethyl sulfanylthiocarbonyl) sulfanylvpentanoic acid (ECT)precursor was synthesized as shown in FIG. 15. The pyridyl disulfidefunctionalized RAFT chain transfer agent (CTA) was synthesized by firstconverting ECT to the NHS ester followed by reaction withpyridyldithio-ethylamine. ECT (1.05 g, 4 mmol) and N-hydroxysuccinimide(0.460 g, 4 mmol) were dissolved in 100 mL of chloroform. The mixturewas then cooled to 0° C. at which time N,N′ dicyclohexylcarbodiimide(0.865 mg, 4.2 mmol) was added. The solution was maintained at 0° C. for1 hour and then allowed to react at room temperature for 22 hours. Thesolution was then filtered to remove the dicyclohexyl urea and thesolution concentrated via rotary evaporation. The resultant solid wasthen dried under vacuum and used without any further purification. NHSECT (1.80 g, 5.0 mmol) and pyridyldithio-ethylamine (0.90 g, 5.0 mmoL)where then separately dissolved in 200 and 300 mL of chloroform,respectively. The solution of pyridyldithio-ethylamine was then addeddropwise as three fractions 20 minutes apart. The mixture was thenallowed to react at room temperature for 2 hours. After solvent removal,two successive column chromatographies (Silica gel 60, Merk) wereperformed (ethyl acetate:hexane 50:50; ethyl acetate:hexane 70:30 v/v)yielding a viscous orange solid. 1H NMR 200 MHz (CDCl3, RT, ppm)1.29-1.41 [t, CH3CH2S: 3H], 1.85-1.93 [s, (CH₃)C(CN): 3H], 2.33-2.59 [m,C(CH₃)(CN)(CH₂CH₂): 4H], 2.86-2.97 [t, CH₂SS: 2H], 3.50-3.61 [t, NHCH₂:2H], 7.11-7.22 [m, Ar Para CH: 1H], 7.46-7.52 [m, Ar CH Ortho: 1H],7.53-7.62 [br, NH: 1H], 7.53-7.68 [m, Ar meta CH: 1H], 8.47-8.60 [m,meta CHN, 1H]. [00164] Preparation of Thiol Reactive Polymer: RAFTPolymerization of Pyridyl Disulfide Functionalizedpoly[HPMA]-b-[(PAA)(BMA)(DMAEMA)]. The RAFT polymerization ofN-(2-hydroxypropyl) methacrylamide (HPMA) was conducted in methanol (50weight percent monomer:solvent) at 70° C. under a nitrogen atmospherefor 8 hours using 2,2′-azo-bis-isobutyrylnitrile (AIBN) as the freeradical initiator. The molar ratio of CTA to AIBN was 10 to 1 and themonomer to CTA ratio was set so that a molecular weight of 25,000 g/molwould be achieved if at 100% conversion. The poly(HPMA) macro-CTA wasisolated by repeated precipitation into diethyl ether from methanol.

The macro-CTA was dried under vacuum for 24 hours and then used forblock copolymerization of dimethylaminoethyl methacrylate (DMAEMA),propylacrylic acid (PAA), and butyl methacrylate (BMA). Equimolarquantities of DMAEMA, PAA, and BMA ([M]_(o)/[CTA]_(o)=250) were added tothe HPMA macroCTA dissolved in N,N-dimethylformamide (25 wt % monomerand macroCTA to solvent). The radical initiator V70 was added with a CTAto initiator ratio of 10 to 1. The polymerization was allowed to proceedunder a nitrogen atmosphere for 18 hours at 30° C. Afterwards, theresultant diblock polymer was isolated by precipitation 4 times into50:50 diethyl ether/pentane, redissolving in ethanol betweenprecipitations. The product was then washed 1 time with diethyl etherand dried overnight in vacuo.

Gel permeation chromatography (GPC) was used to determine molecularweight and polydispersity (Mw/Mn, PDI) of both the poly(HPMA) macroCTAand the diblock copolymer in DMF. Molecular weight calculations werebased on column elution times relative to polymethyl methacrylatestandards using HPLC-grade DMF containing 0.1 wt % LiBr at 60° C. as themobile phase. Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) wasused to reduce the polymer end pyridyl disulfide, releasing2-pyridinethione. Based on the experimentally determined polymermolecular weight and the molar extinction coefficient of2-pyridinethione at 343 nm (8080 M⁻¹ cm⁻¹) in aqueous solvents, percentend group preservation was determined for the poly(HPMA) macroCTA andthe diblock copolymer.

Example 11. Polymer-Peptide Conjugation

Fusion with the peptide transduction domain peptide transportin (alsoknown as the Antennapedia peptide (Antp) sequence was utilized tosynthesize a cell internalizing form of the Bak BH3 peptide (Antp-BH3)containing a carboxy-terminal cysteine residue(NH2-RQIKIWFQNRRMKWKKMGQVGRQLAIIGDDINRRYDSC-COOH) [ SEQ ID NO: 1]. Toensure free thiols for conjugation, the peptide was reconstituted inwater and treated for 1 hour with the disulfide reducing agent TCEPimmobilized within an agarose gel. The reduced peptide (400 μM) was thenreacted for 24 hours with the pyridyl disulfide end-functionalizedpolymer in phosphate buffer (pH 7) containing 5 mMethylenediaminetetraacetic acid (EDTA).

As shown in FIG. 16, reaction of the pyridyl disulfide polymer end groupwith the peptide cysteine creates 2-pyridinethione, which can bespectrophotometrically measured to characterize conjugation efficiency.To further validate disulfide exchange, the conjugates were run on anSDS PAGE 16.5% tricine gel. In parallel, aliquots of the conjugationreactions were treated with immobilized TCEP prior to SDS-PAGE to verifyrelease of the peptide from the polymer in a reducing environment.

Conjugation reactions were conducted at polymer/peptide stoichiometriesof 1, 2, and 5. UV spectrophotometric absorbance measurements at 343 nmfor 2-pyridinethione release indicated conjugation efficiencies of 40%,75%, and 80%, respectively (moles 2-pyridinethione/moles peptide). AnSDS PAGE gel was utilized to further characterize peptide-polymerconjugates (FIG. 17). At a polymer/peptide molar ratio of 1, adetectable quantity of the peptide formed dimers via disulfide bridgingthrough the terminal cysteine. However, the thiol reaction to thepyridyl disulfide was favored, and the free peptide band was no longervisible at polymer/peptide ratios equal to or greater than 2 (FIG. 17A). By treating the conjugates with the reducing agent TCEP, it waspossible to cleave the polymer-peptide disulfide linkages as indicatedby the appearance of the peptide band in these samples (FIG. 17 B).

Example 12. pH-Dependent Membrane Destabilizing Properties ofPoly[HPMA]-B-[(PAA)(BMA)(DMAEMA)]

In order to assess the polymer's potential for endosomolytic activity, amembrane disruption assay was utilized to measure the capacity of thepolymer to trigger pH-dependent disruption of lipid bilayer membranes asshown in FIG. 18. Whole human blood was drawn and centrifuged for plasmaremoval. The remaining erythrocytes were washed three times with 150 mMNaCl and resuspended into phosphate buffers corresponding tophysiological (pH 7.4), early endosome (pH 6.6), and late endosome (pH5.8) environments. The polymer (1-40 μg/mL) or 1% Triton X-100 was addedto the erythrocyte suspensions and incubated for 1 hour at 37° C. Intacterythrocytes were pelleted via centrifugation, and the hemoglobincontent within the supernatant was measured via absorbance at 541 nm.Percent hemolysis was determined relative to Triton X-100. Polymerhemolysis was quantified at concentrations ranging from 1-40 μg/mLrelative to 1% v/v Triton X-100. This experiment was completed 2 timesin triplicate, yielding similar results. The data shown represent asingle experiment conducted in triplicate±standard deviation.

Red blood cell hemolysis measures pH-dependent membrane disruptionproperties of the diblock copolymer at pH values mimicking physiologic(7.4), early endosomal (6.6) and late endosomal (5.8) environments. Atphysiologic pH, no significant red blood cell membrane disruption wasobserved even at polymer concentrations as high as 40 μg/mL (FIG. 18).However, as the pH was lowered to endosomal values, a significantincrease in hemolysis was detected, with greater membrane disruption atpH 5.8 compared to 6.6. The hemolytic behavior of the polymer correlatedto polymer concentration, with nearly 70% erythrocyte lysis occurring at40 μg/mL polymer in pH 5.8 buffer. This sharp “switch” to a membranedestabilizing conformation at endosomal pH combined with negligiblemembrane activity in the physiologic pH range indicates potential forthis polymer as a non-toxic intracellular delivery vehicle.

Example 13. Characterization of Intracellular Delivery in Hela Cells

HeLas, human cervical carcinoma cells (ATCC CCL-2), were maintained inminimum essential media (MEM) containing L-glutamine, 1%penicillin-streptomycin, and 10% FBS. Prior to experiments, HeLas wereallowed to adhere overnight in 8-well chamber slides (20,000 cells/well)for microscopy or 96-well plates (10,000 cells/well) for other assays.Polymer-peptide conjugates and controls were added in MEM with 1% FBS.

Polymer intracellular delivery potential was evaluated followingbioconjugation to the Bak-BH3 peptide fused with the Antp (penetratin)cell penetrating peptide. BH3 fusion to Antp has been extensivelystudied as a cell translocation domain and has previously been found totrigger apoptotic signaling (Li et al. Neoplasia (New York, N.Y. 2007;9(10):801-811). However, it is believed that therapeutics delivered viapeptidic transduction domains may suffer from hindered potency due tosequestration within intracellular vesicles (Sugita et al. BritishJournal of Pharmacology. 2008; 153(6):1143-1152). The following in vitrostudies demonstrate that the combined Antp-BH3 peptide cytoplasmicdelivery and pro-apoptotic functionality was enhanced by conjugation tothe diblock polymer.

Microscopic Analysis of Conjugate Endosomal Escape. An amine reactiveAlexa-488 succinimidyl ester was mixed at a 1 to 1 molar ratio with theAntp-BH3 peptide in anhydrous dimethyl formamide (DMF). Unreactedfluorophore and organic solvent were removed using a PD10 desaltingcolumn, and the fluorescently labeled peptide was lyophilized. Alexa-488labeled Antp-BH3 was conjugated to the polymer as described above. Freepeptide or polymer-peptide conjugate was applied to HeLas grown onchambered microscope slides at a concentration of 25 μM Antp-BH3. Cellswere treated for 15 minutes, washed twice with PBS, and incubated infresh media for an additional 30 minutes. The samples were washed againand fixed with 4% paraformaldehyde for 10 minutes at 37° C. Slides weremounted with ProLong Gold Antifade reagent containing DAPI and imagedusing a fluorescent microscope.

To study the effects of polymer conjugation on peptide endosomal escape,the Alexa-488 labeled peptide was analyzed by fluorescent microscopy.The fluorescently labeled peptide was delivered alone or as the polymerbioconjugate. Microscopic analysis revealed clear differences in peptideintracellular localization following polymer conjugation (FIG. 19). Thepeptide alone displayed punctate staining, indicative of endosomalcompartmentalization. Samples delivered polymer-peptide conjugateexhibited a dispersed fluorescence pattern, consistent with peptidediffusion throughout the cytoplasm. Representative images illustrating(FIG. 19A) punctate peptide staining (green) in the samples deliveredpeptide alone and (FIG. 19B) dispersed peptide fluorescence within thecytosol following delivery of peptide-polymer conjugate. Samples weretreated for 15 minutes with 25 μM peptide and prepared for microscopicexamination following DAPI nuclear staining (blue).

Measurement of Conjugate Cytotoxicity. Bioconjugate efficacy fortriggering tumor cell death was determined using a lactate dehydrogenase(LDH) cytotoxicity assay. At the end of each time point, cells werewashed two times with PBS and then lysed with cell lysis buffer (100μL/well, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA,1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mMsodium orthovanadate) for 1 hour at 4° C. 20 μl of lysate from eachsample was diluted into 80 μl PBS, and LDH was quantified by mixing with100 μL of the LDH substrate solution. Following a 10 minute incubation,LDH was determined by measuring absorbance at 490 nm. Percent viabilitywas expressed relative to samples receiving no treatment.

To assess polymer-peptide conjugate bioactivity, a cytotoxicity studywas conducted in HeLa cervical cancer cells. The Antp-BH3 polymerconjugate was found to potently trigger HeLa cell death in a dosedependent fashion. Less than 50% HeLa viability was detected after 6hours of treatment with 10 μM peptide conjugate (FIG. 20A), and samplesreceiving 25 μM peptide conjugate (FIG. 20B) showed little if any viablecells following as little as 4 hours of exposure. Control samplesreceiving peptide or polymer alone displayed negligible treatmenteffect, and there was no difference between these control treatmentgroups. Importantly, Antp-BH3 poly(HPMA) conjugates that lacked thepH-responsive block were similar to both control groups and did notresult in significant toxicity, further validating the functionality ofthe endosomolytic block (FIG. 20).

Flow Cytometry Evaluation of Mitochondrial Membrane Potential. Loss ofmitochondrial membrane potential, a known indicator for apoptosis, wasassessed using the JC-1 dye. JC-1 exhibits green fluorescence whendispersed in the cytosol, and in healthy cells, it forms red-fluorescentaggregates at the mitochondrial membrane (Cossarizza et al. Biochemicaland biophysical research communications. 1993; 197(1):40-45). HeLas wereincubated for 2 hours with 10 μM peptide or equivalent conjugate orpolymer alone. JC-1 was added at a final concentration of 5 μg/mL andincubated for 15 minutes. Cells were washed 2 times with PBS,trypsinized, and resuspended in 0.5% BSA for flow cytometric analysis.Percent of cells displaying mitochondrial depolarization was quantifiedbased on the number of green fluorescent cells that were negative forred fluorescence. Here, a significant loss of red fluorescent JC-1aggregates and therefore a loss in mitochondrial polarization wasdetected following treatment with both the Antp-BH3 peptide and thepolymer peptide conjugate (FIG. 21 A). Polymer controls were similar tocells receiving no treatment while Antp-BH3 alone and in a polymerconjugate resulted in an approximately 4- and 10-fold increase,respectively, in percent of cells exhibiting loss of mitochondrialpolarity.

Caspase 3/7 Activity Assay. Caspase 3/7 activation was measured using acommercially available assay kit. This assay utilizes a profluorescentcaspase 3/7 substrate that once enzymatically cleaved becomesfluorescent allowing for determination of relative enzyme activity usinga fluorescent plate reader. Here, HeLas were incubated for 30 minuteswith 25 μM peptide (alone or as polymer conjugate) in addition topolymer alone in a quantity equivalent to the conjugate samples.Afterwards, a caspase 3/7 fluorigenic indicator was added directly tothe culture media for each sample. Plates were shaken for 1 hour andthen assayed using a fluorescent plate reader. Data were expressed aspercent caspase activity relative to samples receiving no treatment.

Activation of caspases 3 and 7, which is indicative of pro-apoptoticsignaling, can be measured using a profluorescent substrate specific tothese proteases. FIG. 21 B shows that controls containing the polymeralone displayed equivalent caspase activity relative to negativecontrols receiving no treatment. However, rapid caspase activation(approximately 2.5-fold) was detected following treatment with theAntp-BH3 peptide by itself or in the polymer conjugate form. The similareffects of Antp-BH3 alone or as a polymer conjugate could indicate thatcaspase signaling is saturated by treatment with the peptide alone orthat other positive feedback mechanisms exist for amplification ofperturbations in caspase activation state. Minimally, these resultssuggest that there was no steric hindrance or other reductions inpeptide-induced caspase activity as a result of conjugation to thepolymer.

1-39. (canceled)
 40. An endosomolytic copolymer comprising: (a) a firstblock, the first block being a hydrophilic block comprising a firstchargeable species that is cationic at about neutral pH; (b) a secondblock, the second block being a membrane destabilizing hydrophobic blockcomprising: (i) a second chargeable species that is anionic at aboutneutral pH; and (ii) a third chargeable species that is cationic atabout neutral pH; and (c) an oligonucleotide associated with the firstblock; wherein the first chargeable species is present on a firstconstitutional unit, the second chargeable species is present on asecond constitutional unit, and the third chargeable species is presenton a third constitutional unit.
 41. The copolymer of claim 40, whereinthe copolymer is a diblock copolymer.
 42. The copolymer of claim 40,wherein the first chargeable species is selected from the groupconsisting of amino, alkylamino, guanidine, imidazole, and pyridyl. 43.The copolymer of claim 42, wherein the first constitutional unit is aresidue of dialkylaminoalkylmethacrylate (DMAEMA).
 44. The copolymer ofclaim 40, wherein the second chargeable species is selected from thegroup consisting of carboxylate, boronate, phosphonate, and phosphate.45. The copolymer of claim 44, wherein the second constitutional unit isa residue of ethyl acrylic acid or propyl acrylic acid.
 46. Thecopolymer of claim 40, wherein the third chargeable species is selectedfrom the group consisting of amino, alkylamino, guanidine, imidazole,and pyridyl.
 47. The copolymer of claim 46, wherein the thirdconstitutional unit is a residue of dialkylaminoalkylmethacrylate(DMAEMA).
 48. The copolymer of claim 40, wherein the secondconstitutional unit is a residue of propyl acrylic acid (PAA) and thethird constitutional unit is a residue of dialkylaminoalkylmethacrylate(DMAEMA), and wherein the second block further comprises a residue ofbutyl methacrylate (BMA) as a fourth constitutional unit.
 49. Thecopolymer of claim 40, comprising the chemical Formula II:

wherein A₀, A₁, A₂, A₃ and A₄ are selected from the group consisting of—C—C—, —C(O)(C)_(a)C(O)O—, and —O(C)_(a)C(O)—, wherein a is 1-4; Y₀ andY₄ are independently selected from the group consisting of hydrogen,-(1C-10C)alkyl, -(3C-6C)cycloalkyl, —O-(1C-10C)alkyl,—C(O)O(1C-10C)alkyl, —C(O)NR₆(1C-10C), -(4C-10C)hetroaryl and-(6C-10C)aryl, any of which is optionally substituted with one or morefluorine groups; Y₁ and Y₂ are independently selected from the groupconsisting of a covalent bond, -(1C-10C)alkyl-, —C(O)O(2C-10C)alkyl-,—OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl-, —S(2C-10C)alkyl-,—C(O)NR₆(2C-10C)alkyl-, -(4C-10C)hetroaryl- and -(6C-10C)aryl-; Y₃ isselected from the group consisting of a covalent bond, -(1C-10C)alkyl-,-(4C-10C)hetroaryl- and -(6C-10C)aryl-; wherein tetravalent carbon atomsof A₀-A₄ that are not fully substituted with R₁-R₅ and Y₀—Y₄ arecompleted with an appropriate number of hydrogen atoms; R₁, R₂, R₃, R₄,R₅, and R₆ are independently selected from the group consisting ofhydrogen, —CN, alkyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl, any of which may be optionallysubstituted with one or more fluorine atoms; Q₁ and Q₂ are residueswhich are positively charged at normal physiological pH; Q₃ is a residuewhich is negatively charged at normal physiological pH, but undergoesprotonation at lower pH; m is a mole fraction of 0 to 0.49; n is a molefraction of 0.51 to 1.0; wherein m+n=1 p is a mole fraction of 0.2 to0.5; q is a mole fraction of 0.2 to 0.5; wherein p is substantially thesame as q; r is a mole fraction of 0 to 0.6; wherein p+q+r=1 v is from 1to 25 kDa; and, w is from 1 to 50 kDa.
 50. The copolymer of claim 49,wherein m is
 0. 51. The copolymer of claim 49, wherein R₂-A₁-Y₁-Q₁ is aresidue of a C1-6 dialkylamino(C1-6)alkylmethacrylate, C1-6alkylamino(C1-6)alkylmethacrylate, amino(C1-6)alkylacrylate, C1-6dialkylamino(C1-6)alkylethacrylate, C1-6alkylamino(C1-6)alkylethacrylate, amino(C1-6)alkylethacrylate, C1-6dialkylamino(C1-6)alkylacrylate, C1-6 alkyl amino(C1-6)alkylacrylate, oramino(C1-6)alkylacrylate.
 52. The copolymer of claim 51, whereinR₂-A₁-Y₁-Q₁ is a residue of dialkylaminoalkylmethacrylate (DMAEMA). 53.The copolymer of claim 49, wherein R₃-A₂-Y₂-Q₂ is a residue of a C1-6dialkylamino(C1-6)alkylmethacrylate, C1-6alkylamino(C1-6)alkylmethacrylate, amino(C1-6)alkylacrylate, C1-6dialkylamino(C1-6)alkylethacrylate, C1-6alkylamino(C1-6)alkylethacrylate, amino(C1-6)alkylethacrylate, C1-6dialkylamino(C1-6)alkylacrylate, C1-6 alkyl amino(C1-6)alkylacrylate, oramino(C1-6)alkylacrylate.
 54. The copolymer of claim 53, whereinR₃-A₂-Y₂-Q₂ is a residue of dialkylaminoalkylmethacrylate (DMAEMA). 55.The copolymer of claim 49, wherein R₄-A₃-Y₃-Q₃ is a residue of ethylacrylic acid or propyl acrylic acid.
 56. The copolymer of claim 49,wherein R₅-A₄-Y₄ is a residue of a C1-6 alkylacrylate, C1-C6alkylmethacrylate, or C1-C6 alkylethacrylate.
 57. The copolymer of claim49, comprising the chemical Formula IV3 or Formula IV5:[DMAEMA]_(v)-[B_(p)-/-P_(q)-/-D_(r)]_(w)  (IV3)[PEGMA_(m)-/-DMAEMA_(n)]_(v)-[B_(p)-/-P_(q)-/-Dr]_(w)  (IV5) wherein Bis butyl methacrylate residue; P is propyl acrylic acid residue; D andDMAEMA are dimethylaminoethyl methacrylate residue; and PEGMA ispolyethyleneglycol methacrylate residue.
 58. The copolymer of claim 40,wherein the copolymer comprises a targeting moiety.
 59. The copolymer ofclaim 40, wherein the oligonucleotide is selected from the groupconsisting of an siRNA, a microRNA (miRNA), a short hairpin RNA (shRNA),an asymmetrical interfering RNA (aiRNA), and a dicer substrate.